Modified oligonucleotides targeting SNPs

ABSTRACT

Novel oligonucleotides that enhance silencing of the expression of a gene containing a single nucleotide polymorphism (SNP) relative to the expression of the corresponding wild-type gene are provided. Methods of using novel oligonucleotides that enhance silencing of the expression of a gene containing a SNP relative to the expression of the corresponding wild-type gene are provided.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/717,287 filed Aug. 10, 2018, and 62/825,429 filed Mar. 28, 2019.The entire contents of these applications are incorporated herein byreference.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbersNS104022 and GM108803 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 27, 2022, isnamed 614776_UM9-227_ST25.txt and is 90,752 bytes in size.

BACKGROUND

RNA interference represents a simple and effective tool for inhibitingthe function of genes. RNA silencing agents have received particularinterest as research tools and therapeutic agents for their ability toknock down expression of a particular protein with a high degree ofsequence specificity. The sequence specificity of RNA silencing agentsis particularly useful for the treatment of diseases caused by dominantmutations in heterozygotes bearing one mutant and one wild-type copy ofa particular gene. However, there remains a need for RNA silencingagents that can preferentially silence mutant, disease-causing alleleexpression while not or only minimally effecting expression of thewild-type allele.

SUMMARY

The present invention is based on the surprising discovery of noveloligonucleotides that enhance silencing of the expression of a genecontaining a single nucleotide polymorphism (SNP) (e.g., a heterozygousSNP) relative to the expression of the corresponding wild-type gene in aheterozygote, e.g., by up to more than 100 times. In certain aspects, anoligonucleotide (e.g., a dsRNA) is provided that preferentially targetsa SNP-containing nucleic acid for degradation, wherein theoligonucleotide (e.g., a double-stranded RNA (dsRNA)) does not target,or targets to a lesser degree, the corresponding wild-type(non-SNP-containing) nucleic acid for degradation. In certain aspects,an oligonucleotide (e.g., a dsRNA) of the invention is: 1) complementaryto a SNP position in a target nucleic acid; and 2) contains a mismatchat a particular position of the target nucleic acid relative to the SNP.In certain embodiments, an oligonucleotide (e.g., a dsRNA) of theinvention also contains a two mismatches relative to the correspondingwild-type target nucleic acid sequence: 1) at the wild-type SNPposition; and 2) at the particular position of the target nucleic acidsequence relative to the wild-type SNP position. Accordingly, anexemplary oligonucleotide (e.g., dsRNA) contains one mismatch relativeto a SNP-containing target and two mismatches relative to thecorresponding wild-type sequence, thus resulting in preferentialcleavage of the SNP-containing target relative to the correspondingwild-type sequence.

In one aspect, an oligonucleotide having a 5′ end, a 3′ end and a seedregion, wherein the RNA is complementary to a region of a genecomprising an allelic polymorphism, and wherein the RNA comprises a SNPposition nucleotide at a position within the seed region, wherein theSNP position nucleotide is complementary to the allelic polymorphism;and a mismatch (MM) position nucleotide located 2-11 nucleotides fromthe SNP position nucleotide that is a mismatch with a nucleotide in thegene is provided. In some cases, the oligonucleotide is complementary toa region of a gene comprising an allelic polymorphism, wherein theoligonucleotide comprises an SNP position nucleotide at any one ofpositions 2 to 6 from the 5′ end; and a mismatch position nucleotidelocated 2-11 nucleotides from the SNP position nucleotide that is amismatch with a nucleotide in the gene.

In certain exemplary embodiments, the oligonucleotide is RNA.

In certain exemplary embodiments, the RNA further comprises at least onevinyl phosphonate (VP) modification in an intersubunit linkage havingthe formula:

In certain exemplary embodiments, a VP motif is inserted next to the SNPposition nucleotide or next to the MM position nucleotide.

In certain exemplary embodiments, oligonucleotide is selected from thegroup consisting of siRNA, miRNA, shRNA, CRISPR guide, DNA, antisenseoligonucleotide (ASO), gapmer, mixmer, miRNA inhibitor, splice-switchingoligonucleotide (SSO), phosphorodiamidate morpholino oligomer (PMO), andpeptide nucleic acid (PNA).

In certain exemplary embodiments, the RNA is an antisenseoligonucleotide (ASO) or a dsRNA.

In certain exemplary embodiments, the dsRNA comprises a first strand ofabout 15-35 nucleotides that is complementary to the region of the genecomprising an allelic polymorphism, and a second strand of about 15-35nucleotides that is complementary to at least a portion of the firststrand, wherein the first strand comprises the SNP position nucleotidein a seed region (e.g., at position 2-6 from the 5′ end) that iscomplementary to the allelic polymorphism, and wherein the first strandcomprises the MM position nucleotide located 2-6 nucleotides from theSNP position nucleotide that is a mismatch with a nucleotide in thegene.

In certain exemplary embodiments, the SNP position nucleotide is locatedat position 2, 4 or 6 from the 5′ end of the RNA, and the MM positionnucleotide is located 2-6 nucleotides from the SNP position nucleotide.

In certain exemplary embodiments, the MM position nucleotide is locatedwithin 2, 3, 4 or 6 nucleotides of the SNP position nucleotide. Incertain exemplary embodiments, the MM position nucleotide is locatedwithin 5 nucleotides of the SNP position nucleotide.

In certain exemplary embodiments, the dsRNA is blunt-ended. In certainexemplary embodiments, the dsRNA comprises at least one single-strandednucleotide overhang. In certain exemplary embodiments, the dsRNAcomprises naturally occurring nucleotides. In certain exemplaryembodiments, the dsRNA comprises at least one modified nucleotide. Incertain exemplary embodiments, each nucleotide of the dsRNA is modified.

In certain exemplary embodiments, the at least one modified nucleotideis selected from the group consisting of a 2′-O-methyl modifiednucleotide, a nucleotide comprising a 5′-phosphorothioate group, aterminal nucleotide linked to a cholesteryl derivative and a terminalnucleotide linked to a dodecanoic acid bisdecylamide group.

In certain exemplary embodiments, the modified nucleotide is selectedfrom the group consisting of a 2′-deoxy-2′-fluoro modified nucleotide, a2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide,a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, amorpholino nucleotide, a phosphoramidate, and a non-natural basecomprising nucleotide.

In certain exemplary embodiments, the dsRNA comprises at least one2′-O-methyl modified nucleotide and a 5′-phosphorothioate group.

In certain exemplary embodiments, the first strand comprises at leastthree 2′-O-methyl modified nucleotides. In certain exemplaryembodiments, the first strand comprises a 2′-O-methyl modifiednucleotide on either side of the SNP position nucleotide.

In certain exemplary embodiments, the dsRNA comprises a hydrophobicmoiety.

In certain exemplary embodiments, the region of a gene comprising theallelic polymorphism comprises a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 1-10. In certain exemplary embodiments,the first strand comprises UUCUGUAGCAUCAGCUUCUC

In certain exemplary embodiments, an RNA described herein (e.g., thefirst strand of a dsRNA) comprises a SNP position nucleotide (referencedfrom the 5′ end)—MM position nucleotide (referenced from the 5′ end)combination selected from the group consisting of 2-7, 4-7, 4-8, 4-15,6-5, 6-8, 6-11, 6-14, 6-16, 3-5, 3-7 and 3-8.

In certain exemplary embodiments, the SNP position nucleotide iscomplementary to an allelic polymorphism of an htt SNP selected from thegroup consisting of rs363125, rs362273, rs362307, rs362336, rs362331,rs362272, rs362306, rs362268, rs362267, and rs363099.

In certain exemplary embodiments, the RNA further comprises a 5′stabilizing moiety selected from the group consisting of phosphate,vinyl phosphonate, C5-methyl (R or S or racemic), C5-methyl on vinyl,and reduced vinyl.

In certain exemplary embodiments, the RNA further comprises a conjugatemoiety selected from the group consisting of alkyl chain, vitamin,peptide, glycosphingolipid, polyunsaturated fatty acid, secosteroid,steroid hormone, and steroid lipid.

In one aspect, a dsRNA comprising a first strand of about 15-35nucleotides that is complementary to a region of a gene comprising anallelic polymorphism, and a second strand of about 15-35 nucleotidesthat is complementary to at least a portion of the first strand, whereinthe first strand comprises a SNP position nucleotide at any one ofpositions in the seed region (e.g., one of positions 2 to 6 from the 5′end) that is complementary to the allelic polymorphism, and wherein thefirst strand comprises a MM position nucleotide located 2-6 nucleotidesfrom the SNP position nucleotide that is a mismatch with a nucleotide inthe gene, is provided.

In certain exemplary embodiments, a dsRNA comprising a first strand ofabout 15-35 nucleotides that is complementary to a region of a genecomprising an allelic polymorphism, and a second strand of about 15-35nucleotides that is complementary to at least a portion of the firststrand, wherein the first strand comprises a SNP position nucleotide atposition 2, 4 or 6 from the 5′ end that is complementary to the allelicpolymorphism, and wherein the first strand comprises a MM positionnucleotide located 2-6 nucleotides from the SNP position nucleotide thatis a mismatch with a nucleotide in the gene, is provided.

In certain exemplary embodiments, the RNA further comprises at least onevinyl phosphonate modification in an intersubunit linkage having theformula:

In certain exemplary embodiments, a VP motif is inserted next to the SNPposition nucleotide or next to the MM position nucleotide.

In certain exemplary embodiments, the SNP position nucleotide is atposition 2 from the 5′ end, at position 4 from the 5′ end, or atposition 6 from the 5′ end of the first strand. In other exemplaryembodiments, the MM position nucleotide is located within 2 nucleotidesof the SNP position nucleotide, is located within 3 nucleotides of theSNP position nucleotide, is located within 4 nucleotides of the SNPposition nucleotide, is located within 5 nucleotides of the SNP positionnucleotide, or is located within 6 nucleotides of the SNP positionnucleotide. In certain exemplary embodiments, the SNP positionnucleotide is located 4 nucleotides from the 5′ end, and the MM positionnucleotide is located 7 nucleotides from the 5′ end. In other exemplaryembodiments, the SNP position nucleotide is located 6 nucleotides fromthe 5′ end, and the MM position nucleotide is located 11 nucleotidesfrom the 5′ end.

In another aspect, a dsRNA comprising a first strand of about 15-35nucleotides that is complementary to a region of a gene comprising anallelic polymorphism, and a second strand of about 15-35 nucleotidesthat is complementary to at least a portion of the first strand, whereinthe first strand comprises a SNP position nucleotide at a position 6from the 5′ end that is complementary to the allelic polymorphism, andwherein the first strand comprises a MM position nucleotide located atposition 11 from the 5′ end is a mismatch with a nucleotide in the gene,is provided.

In certain exemplary embodiments, the RNA further comprises at least onevinyl phosphonate modification in an intersubunit linkage having theformula:

In certain exemplary embodiments, a VP motif is inserted next to the SNPposition nucleotide or next to the MM position nucleotide.

In certain exemplary embodiments, the first strand comprises a2′-O-methyl modified nucleotide on either side of the SNP positionnucleotide. In certain exemplary embodiments, the first strand comprisesat least three 2′-O-methyl modified nucleotides.

In certain exemplary embodiments, the dsRNA comprises a5′-phosphorothioate group.

In certain exemplary embodiments, the gene comprising an allelicpolymorphism is the Huntingtin (htt) gene.

In certain exemplary embodiments, the region of a gene comprising theallelic polymorphism comprises a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 1-10. In certain exemplary embodiments,the first strand comprises UUCUGUAGCAUCAGCUUCUC

In certain aspects, a pharmaceutical composition comprising the RNAdescribed herein and a pharmaceutically acceptable carrier is provided.

In certain aspects, a method of inhibiting expression of a genecomprising an allelic polymorphism in a cell, the method comprisingcontacting the cell with the described herein is provided.

In another aspect, a method of treating a disease or disordercharacterized or caused by a gene comprising an allelic polymorphism ina subject in need thereof, comprising administering to a subject atherapeutically effective amount of an RNA having a 5′ end, a 3′ end,and a seed region, that is complementary to a region of a genecomprising an allelic polymorphism, wherein the RNA comprises a SNPposition nucleotide at position in the seed region that is complementaryto the allelic polymorphism, and a MM position nucleotide located 2-11nucleotides from the SNP position nucleotide that is a mismatch with anucleotide in the gene, provided.

In certain exemplary embodiments, the RNA further comprises at least onevinyl phosphonate modification in an intersubunit linkage having theformula:

In certain exemplary embodiments, a VP motif is inserted next to the SNPposition nucleotide or next to the MM position nucleotide.

In certain exemplary embodiments, the RNA is an ASO or a dsRNA.

In certain exemplary embodiments, the dsRNA comprises a first strand ofabout 15-35 nucleotides that is complementary to the region of the genecomprising an allelic polymorphism, and a second strand of about 15-35nucleotides that is complementary to at least a portion of the firststrand, wherein the first strand comprises the SNP position nucleotidewithin the seed region (e.g, any one of positions 2 to 6 from the 5′end, such as position 2, 4 or 6 from the 5′ end) that is complementaryto the allelic polymorphism, and wherein the first strand comprises theMM position nucleotide located 2-6 nucleotides from the SNP positionnucleotide that is a mismatch with a nucleotide in the gene.

In certain exemplary embodiments, said dsRNA is administered to thebrain of the subject. In certain exemplary embodiments, said dsRNA isadministered by intrastriatal infusion. In certain exemplaryembodiments, a decrease in expression of the gene in the striatum isachieved. In certain exemplary embodiments, a decrease in expression ofthe gene in the cortex is achieved.

In certain exemplary embodiments, the gene comprising an allelicpolymorphism is the Huntingtin (htt) gene. In certain exemplaryembodiments, the disease is Huntington's disease.

In certain exemplary embodiments, the SNP position nucleotide is locatedat position 2, 4 or 6 from the 5′ end of the RNA, and the MM positionnucleotide is located 2-6 nucleotides from the SNP position nucleotide.

In another aspect, a di-branched oligonucleotide compound comprising twoRNAs, wherein the RNAs are connected to one another by one or moremoieties selected from a linker, a spacer and a branching point, whereineach RNA has a 5′ end, a 3′ end and a seed region, wherein each RNA iscomplementary to a region of a gene comprising an allelic polymorphism,and wherein each RNA comprises a SNP position nucleotide at a positionwithin the seed region, the SNP position nucleotide being complementaryto the allelic polymorphism, and a MM position nucleotide located 2-11nucleotides from the SNP position nucleotide that is a mismatch with anucleotide in the gene, is provided.

In certain exemplary embodiments, the RNA further comprises at least onevinyl phosphonate modification in an intersubunit linkage having theformula:

In certain exemplary embodiments, a VP motif is inserted next to the SNPposition nucleotide or next to the MM position nucleotide.

In certain exemplary embodiments, the di-branched oligonucleotidecompound has an hsi-RNA structure.

In certain exemplary embodiments, the SNP position nucleotide iscomplementary to an allelic polymorphism of an htt SNP selected from thegroup consisting of rs363125, rs362273, rs362307, rs362336, rs362331,rs362272, rs362306, rs362268, rs362267, and rs363099.

In another aspect, a di-branched oligonucleotide compound comprising twoor more nucleic acid sequences, wherein the nucleic acid sequences (N)are connected to one another by one or more moieties selected from alinker (L), a spacer (S) and optionally a branching point (B), whereineach nucleic acid sequence is double-stranded and comprises a sensestrand and an antisense strand, wherein the sense strand and theantisense strand each have a 5′ end and a 3′ end, wherein the sensestrand and the antisense strand each comprises one or morechemically-modified nucleotides, wherein each antisense strand has aseed region, wherein each antisense strand is complementary to a regionof a gene comprising an allelic polymorphism, and wherein each antisensestrand comprises a SNP position nucleotide at a position within the seedregion, the SNP position nucleotide being complementary to the allelicpolymorphism, and a mismatch (MM) position nucleotide located 2-11nucleotides from the SNP position nucleotide that is a mismatch with anucleotide in the gene, is provided.

In certain exemplary embodiments, the RNA further comprises at least onevinyl phosphonate modification in an intersubunit linkage having theformula:

In certain exemplary embodiments, a VP motif is inserted next to the SNPposition nucleotide or next to the MM position nucleotide.

In certain exemplary embodiments, the sense strands and the antisensestrands each comprise >80% chemically-modified nucleotides.

In certain exemplary embodiments, the nucleotides at positions 1 and 2from the 5′ end of the sense and antisense strands are connected toadjacent nucleotides via phosphorothioate linkages.

In certain exemplary embodiments, each antisense strand comprises atleast 15 contiguous nucleotides, and wherein each sense strand comprisesat least 15 contiguous nucleotides and has complementarity to theantisense strand.

In certain exemplary embodiments, the compound further comprises ahydrophobic moiety attached to the terminal 5′ position of the branchedoligonucleotide compound.

In certain exemplary embodiments, each double-stranded nucleic acidsequence is independently connected to a linker, spacer or branchingpoint at the 3′ end or at the 5′ end of the sense strand or theantisense strand.

In certain exemplary embodiments, the SNP position nucleotide iscomplementary to an allelic polymorphism of an htt SNP selected from thegroup consisting of rs363125, rs362273, rs362307, rs362336, rs362331,rs362272, rs362306, rs362268, rs362267, and rs363099.

In another aspect, a nucleic acid having a 5′ end, a 3′ end and a seedregion, that is complementary to a region of a gene comprising anallelic polymorphism, wherein the nucleic acid comprises a SNP positionnucleotide at a position within the seed region, wherein the SNPposition nucleotide is complementary to the allelic polymorphism, a MMposition that is a mismatch with a nucleotide in the gene, and at leastone modified nucleotide (X) on either side of the SNP positionnucleotide, wherein each X is located within four, three or twonucleotides from the SNP position nucleotide, is provided.

In certain exemplary embodiments, the RNA further comprises at least onevinyl phosphonate modification in an intersubunit linkage having theformula:

In certain exemplary embodiments, a VP motif is inserted next to the SNPposition nucleotide or next to the MM position nucleotide.

In certain exemplary embodiments, X comprises a sugar modificationselected from the group consisting of 2′-O-methyl (2′-OMe), 2′-fluoro(2′-F), 2′-ribo, 2′-deoxyribo, 2′-F-4′-thioarabino (2′-F-ANA),2-O-(2-methoxyethyl) (2′-MOE), 4′-S-RNA, locked nucleic acid (LNA),4′-S-F-ANA, 2′-O-allyl, 2′-O-ethylamine, 2-cyanoethyl-RNA (CNet-RNA),tricyclo-DNA, cyclohexenyl nucleic acid (CeNA), arabino nucleic acid(ANA), and hexitol nucleic acid (HNA).

In certain exemplary embodiments, an X is positioned immediately 5′ tothe SNP position nucleotide or immediately 3′ to the SNP positionnucleotide. In certain exemplary embodiments, an X is positionedimmediately 5′ to the SNP position nucleotide and immediately 3′ to theSNP position nucleotide.

In certain exemplary embodiments, the SNP position nucleotide is presentfrom position 2 to position 6 from the 5′ end. In certain exemplaryembodiments, the MM position nucleotide is located 2-11 nucleotides fromthe SNP position nucleotide. In certain exemplary embodiments, the MMposition nucleotide is located 2-6 nucleotides from the SNP positionnucleotide.

In another aspect, a nucleic acid having a 5′ end, a 3′ end and a seedregion, that is complementary to a region of a gene comprising anallelic polymorphism, wherein the nucleic acid comprises a SNP positionnucleotide at a position within the seed region, wherein the SNPposition nucleotide is complementary to the allelic polymorphism, a MMposition that is a mismatch with a nucleotide in the gene, and at leastone modified nucleotide (Y) on either side of the MM positionnucleotide, wherein each Y is located within four, three or twonucleotides from the MM position nucleotide, is provided.

In certain exemplary embodiments, the RNA further comprises at least onevinyl phosphonate modification in an intersubunit linkage having theformula:

In certain exemplary embodiments, a VP motif is inserted next to the SNPposition nucleotide or next to the MM position nucleotide.

In certain exemplary embodiments, a Y is positioned immediately 5′ tothe MM position nucleotide or immediately 3′ to the MM positionnucleotide. In certain exemplary embodiments, a Y is positionedimmediately 5′ to the MM position nucleotide and immediately 3′ to theMM position nucleotide.

In certain exemplary embodiments, the SNP position nucleotide is presentfrom position 2 to position 6 from the 5′ end. In certain exemplaryembodiments, the MM position nucleotide is located 2-11 nucleotides fromthe SNP position nucleotide. In certain exemplary embodiments, the MMposition nucleotide is located 2-6 nucleotides from the SNP positionnucleotide.

In another aspect, a nucleic acid having a 5′ end, a 3′ end and a seedregion, that is complementary to a region of a gene comprising anallelic polymorphism, wherein the nucleic acid comprises a SNP positionnucleotide at a position within the seed region, wherein the SNPposition nucleotide is complementary to the allelic polymorphism, a MMposition that is a mismatch with a nucleotide in the gene, and at leastone modified nucleotide (X) on either side of the SNP positionnucleotide, wherein each X is located within four, three or twonucleotides from the SNP position nucleotide, and at least one modifiednucleotide (Y) on either side of the MM position nucleotide, whereineach Y is located within four, three or two nucleotides from the MMposition nucleotide, is provided.

In certain exemplary embodiments, the RNA further comprises at least onevinyl phosphonate modification in an intersubunit linkage having theformula:

In certain exemplary embodiments, a VP motif is inserted next to the SNPposition nucleotide or next to the MM position nucleotide.

In certain exemplary embodiments, X comprises a sugar modificationselected from the group consisting of 2′-OMe, 2′-F, 2′-ribo,2′-deoxyribo, 2′-F-ANA, 2′-MOE, 4′-S-RNA, LNA, 4′-S-F-ANA, 2′-O-allyl,2′-O-ethylamine, CNet-RNA, tricyclo-DNA, CeNA, ANA, and HNA. In certainexemplary embodiments, Y comprises a sugar modification selected fromthe group consisting of 2′-OMe, 2′-F, 2′-ribo, 2′-deoxyribo, 2′-F-ANA,2′-MOE, 4′-S-RNA, LNA, 4′-S-F-ANA, 2′-O-allyl, 2′-O-ethylamine,CNet-RNA, tricyclo-DNA, CeNA, ANA, and HNA.

In certain exemplary embodiments, an X is positioned immediately 5′ tothe SNP position nucleotide or immediately 3′ to the SNP positionnucleotide. In certain exemplary embodiments, an X is positionedimmediately 5′ to the SNP position nucleotide and immediately 3′ to theSNP position nucleotide.

In certain exemplary embodiments, a Y is positioned immediately 5′ tothe MM position nucleotide or immediately 3′ to the MM positionnucleotide. In certain exemplary embodiments, a Y is positionedimmediately 5′ to the MM position nucleotide and immediately 3′ to theMM position nucleotide.

In certain exemplary embodiments, the SNP position nucleotide is presentfrom position 2 to position 6 from the 5′ end. In certain exemplaryembodiments, the MM position nucleotide is located 2-11 nucleotides fromthe SNP position nucleotide. In certain exemplary embodiments, the MMposition nucleotide is located 2-6 nucleotides from the SNP positionnucleotide.

In certain exemplary embodiments, X and Y are the same.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings.

FIG. 1 depicts psiCHECK reporter plasmids containing either a wild-typeregion of htt or the same region of htt with the SNP, rs362273. FIG. 1discloses SEQ ID NOS 265-268, respectively, in order of appearance.

FIG. 2 depicts a bar graph showing luciferase activity following apsiCHECK reporter plasmid assay in HeLa cells transfected with hsiRNAswith the SNP nucleotide at varying positions. This primary screenyielded multiple efficacious hydrophobically modified siRNA (hsiRNA)sequences.

FIG. 3 depicts dose response curves showing the silencing effects ofthree exemplary hsiRNAs of the invention on psiCHECK reporter plasmids.

FIG. 4 depicts a dose response curve showing the efficacy of two hsiRNAson silencing htt mRNA.

FIG. 5 depicts bar graphs showing luciferase activity following apsiCHECK reporter plasmid assay in HeLa cells transfected with hsiRNAshaving a second mismatch at varying positions.

FIG. 6 depicts dose response curves comparing silencing effects of SNP2hsiRNA with (mm2-7) or without (mm2-0) an additional mismatch.

FIG. 7 depicts dose response curves comparing silencing effects of SNP4hsiRNAs with or without an additional mismatch.

FIG. 8 depicts dose response curves comparing silencing effects of SNP6hsiRNAs with or without an additional mismatch.

FIG. 9 depicts dose response curves comparing silencing effects of SNP4or SNP6 hsiRNAs with an additional mismatch (SNP4-7 and SNP6-11,respectively), compared to the same hsiRNAs without an additionalmismatch (SNP4-0 and SNP4-11). HeLa cells transfected with one of tworeporter plasmids were revers transfected with hsiRNAs by passiveuptake, and treated for 72 hours. reporter expression was measured witha dual luciferase assay.

FIG. 10 depicts dose response curves of htt mRNA expression thatmeasures silencing efficacy of hsiRNAs with additional mismatches.

FIG. 11 schematically depicts an hsiRNA and exemplary modificationsaccording to certain embodiments of the invention.

FIG. 12A-FIG. 12C depict the SNP2, SNP4 and SNP6 hsiRNA libraries,respectively. Antisense strands are depicted 5′ to 3′, with the SNP sitein red and the mismatch in blue. FIG. 12A discloses SEQ ID NOS 269-284,respectively, in order of appearance. FIG. 12B discloses SEQ ID NOS285-300, respectively, in order of appearance. FIG. 12C discloses SEQ IDNOS 301-316, respectively, in order of appearance.

FIG. 13 depicts antisense and sense strand sequences and modificationpatterns for various hsiRNA constructs according to certain embodiments.mm4-7 and mm6-11 demonstrated superior SNP discrimination, and wereselected for further screening. FIG. 13 discloses SEQ ID NOS 317,291-292, 299, 305, 308, 311, 314, 316, 318, 319, 319, 319, 320, 320,320, 320 and 320, respectively, in order of columns.

FIG. 14 depicts an exemplary SNP-selective compound designed as adi-siRNA. FIG. 14 discloses SEQ ID NOS 321-322 and 321-322,respectively, in order of appearance.

FIG. 15 depicts backbone linkages according to certain exemplaryembodiments. Oligonucleotide backbones may comprise one or anycombination of phosphates, phosphorothioates (a racemic mixture orstereospecific), diphosphorothioates, phosphoramidates, peptide nucleicacids (PNAs), boranophosphates, 2′-5′-phosphodiesters, amides,phosphonoacetates, morpholinos and the like

FIG. 16 depicts sugar modifications according to certain exemplaryembodiments. Sugar modifications include one or any combination of2′-O-methyl, 2′-fluoro, 2′-ribo, 2′-deoxyribo, 2′-F-ANA, MOE, 4′-S-RNA,LNA, 4′-S-F-ANA, 2′-O-allyl, 2′-O-ethylamine, CNet-RNA, tricyclo-DNA,CeNA, ANA, HNA and the like.

FIG. 17 depicts internucleotide bonds according to certain exemplaryembodiments. Potential internucleotide bonds can be between the firsttwo nucleotides at the 5′ or 3′ ends of any given oligonucleotide strandcan be stabilized with any of the moieties depicted.

FIG. 18 depicts 5′ stabilization modifications according to certainexemplary embodiments. A suitable 5′ stabilization modification can be aphosphate, no phosphate, a vinyl phosphonate, a C5-methyl (R or S orracemic), a C5-methyl on vinyl, reduced vinyl (e.g., three carbon alkyl)or the like.

FIG. 19 depicts conjugates moieties according to certain exemplaryembodiments. A suitable conjugated moiety can be any length alkyl chain,a vitamin, a ligand, a peptide or a bioactive conjugate, e.g., aglycosphingolipid, a polyunsaturated fatty acid, a secosteroid, asteroid hormone, a steroid lipid, or the like.

FIG. 20 graphically depicts that the activity of a SNP discriminatingscaffold that comprises a SNP position nucleotide at position 6 from the5′ end, and a mismatch position nucleotide located at position 11 fromthe 5′ end, is sequence-independent.

FIG. 21 illustrates a representative synthesis of the vinyl phosphonate(VP)-modified intersubunit linkage described herein.

FIG. 22 depicts a method for preparing oligonucleotides having aVP-modified intersubunit linkage.

FIG. 23 is a pictoral representation of a VP-modified RNA according tocertain exemplary embodiments.

FIG. 24 illustrates the sequences of VP-modified oligonucleotidessynthesized according to certain exemplary embodiments. FIG. 24discloses SEQ ID NOS 323-342, respectively, in order of appearance.

FIG. 25 is a summary of a comparative study of siRNA efficacy.

FIG. 26 is a schematic of hsiRNA antisense scaffolds aligned to HTTsequence surrounding SNP site rs362273 wherein the green box depicts theposition of the SNP site.

FIGS. 27A and 27B illustrate the effect of adding a mismatch in thesiRNA sequence improves allelic discrimination without impairing thesilencing of the mutant allele. FIG. 27A discloses SEQ ID NOS 343-346,234, 347-348, 235, 349-350, 236, 351-352, 237, 353 and 238,respectively, in order of appearance.

FIGS. 28A and 28B depict VP-modified sequences prepared by asynthesizer. FIG. 28A discloses SEQ ID NOS 354-360, respectively, inorder of appearance. FIG. 28B discloses SEQ ID NOS 361-364, 355-360,respectively, in order of appearance.

FIG. 29 demonstrates another method for preparing the VP-modifiedoligonucleotides provided herein.

FIG. 30 demonstrates the effect a VP-modified linkage has ontarget/non-target discrimination of SNP-selective siRNAs.

FIG. 31 illustrates an example di-branched siRNA chemical scaffold.

FIG. 32A is a western blot performed to measure HTT protein levels. FIG.32B shows protein levels normalized to vinculin.

FIG. 33 depicts dose response curves comparing silencing effects foroligonucleotides targeting G at the SNP site instead of A.

FIG. 34 illustrates example sequences introducing single mismatches insequences previously chosen for dose response. FIG. 34 discloses SEQ IDNOS 343-346, 234, 348, 235, 349-350, 236, 351-352, 237, 353, 238,respectively, in order of appearance.

FIG. 35 illustrates a number of exemplary oligonucleotide backbonemodifications. FIG. discloses SEQ ID NOS 365, 386 and 366-373,respectively, in order of columns.

FIG. 36 shows oligonucleotide branching motifs according to certainexemplary embodiments. The double-helices represent oligonucleotides.The combination of different linkers, spacer(s) and branching pointsallows generation of a wide diversity of branched hsiRNA structures.

FIG. 37 shows branched oligonucleotides of the invention with conjugatedbioactive moieties.

FIG. 38 shows exemplary amidite linkers, spacers and branching moieties.

FIG. 39 is a schematic of hsiRNA antisense scaffolds aligned to HTTsequence surrounding alternative SNP site rs362273.

FIG. 40 depicts bar graphs showing luciferase activity following apsiCHECK reporter plasmid assay in HeLa cells transfected with thehsiRNAs of FIG. 39 . The number following “SNP” represents the positionof the SNP in the siRNA.

FIG. 41 depicts dose response curves comparing silencing effects foroligonucleotides of FIG. 39 targeting C or T at the SNP3 site.

FIG. 42 depicts bar graphs showing luciferase activity following apsiCHECK reporter plasmid assay in HeLa cells transfected with hsiRNAsof FIG. 39 which were modified to feature a second mismatch at varyingpositions.

FIG. 43 illustrates example modified intersubunit linkers.

FIG. 44A shows a representative example for preparing a monomer for themodified phosphinate-containing oligonucleotides provided herein. FIG.44B shows a representative example for preparing another monomer for themodified phosphinate-containing oligonucleotides provided herein. FIG.44C shows a representative example for preparing a modifiedphosphinate-containing oligonucleotides provided herein.

FIG. 45 illustrates exemplary SNPs within the HTT gene (SEQ ID NOs: 1-10(numbered from top to bottom)).

FIG. 46 is a flow chart illustrating a methodology for generating andselecting SNP-discriminating siRNAs.

FIG. 47 illustrates a naming convention denoting the position of an SNPwithin an siRNA. FIG. 47 discloses SEQ ID NOS 374-385, respectively, inorder of appearance.

DETAILED DESCRIPTION

The present disclosure relates to compositions comprisingoligonucleotide, e.g., RNA, silencing agents, e.g., RNAs such asdouble-stranded RNAs (“dsRNAs”), antisense oligonucleotides (“ASOs”) andthe like, that are useful for silencing allelic polymorphisms locatedwithin a gene encoding a mutant protein. In a particular aspect, anoligonucleotide, e.g., an RNA, silencing agent is a dsRNA agent providedherein, that destroys a corresponding mutant mRNA (e.g., aSNP-containing mRNA) with nucleotide specificity and selectivity.Oligonucleotide, e.g., RNA, silencing agents, e.g., dsRNA agentsdisclosed herein target mRNA corresponding to polymorphic regions of amutant gene, resulting in cleavage of mutant mRNA, and preventingsynthesis of the corresponding mutant protein e.g., a gain of functionmutant protein such as the huntingtin protein.

Definitions

Unless otherwise defined herein, scientific and technical terms usedherein have the meanings that are commonly understood by those ofordinary skill in the art. In the event of any latent ambiguity,definitions provided herein take precedent over any dictionary orextrinsic definition. Unless otherwise required by context, singularterms shall include pluralities and plural terms shall include thesingular. The use of “or” means “and/or” unless stated otherwise. Theuse of the term “including,” as well as other forms, such as “includes”and “included,” is not limiting.

As used herein in the context of oligonucleotide sequences, “A”represents a nucleoside comprising the base adenine (e.g., adenosine ora chemically-modified derivative thereof), “G” represents a nucleosidecomprising the base guanine (e.g., guanosine or a chemically-modifiedderivative thereof), “U” represents a nucleoside comprising the baseuracil (e.g., uridine or a chemically-modified derivative thereof), and“C” represents a nucleoside comprising the base adenine (e.g., cytidineor a chemically-modified derivative thereof),

As used herein, the term “capping group” refers to a chemical moietythat replaces a hydrogen atom in a functional group such as an alcohol(ROH), a carboxylic acid (RCO₂H), or an amine (RNH₂). Non-limitingexamples of capping groups include: alkyl (e.g., methyl,tertiary-butyl); alkenyl (e.g., vinyl, allyl); carboxyl (e.g., acetyl,benzoyl); carbamoyl; phosphate; and phosphonate (e.g.,vinylphosphonate). Other suitable capping groups are known to those ofskill in the art.

The term “nucleotide analog” or “altered nucleotide” or “modifiednucleotide” refers to a non-standard nucleotide, including non-naturallyoccurring ribonucleotides or deoxyribonucleotides. Exemplary nucleotideanalogs are modified at any position so as to alter certain chemicalproperties of the nucleotide yet retain the ability of the nucleotideanalog to perform its intended function. Examples of positions of thenucleotide which may be derivatized include the 5 position, e.g.,5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine,5-propenyl uridine, and the like; the 6 position, e.g.,6-(2-amino)propyl uridine; the 8-position for adenosine and/orguanosines, e.g., 8-bromo guanosine, 8-chloro guanosine,8-fluoroguanosine, etc. Nucleotide analogs also include deazanucleotides, e.g., 7-deaza-adenosine; O- and N-modified (e.g.,alkylated, e.g., N6-methyl adenosine, or as otherwise known in the art)nucleotides; and other heterocyclically modified nucleotide analogs suchas those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000Aug. 10(4):297-310.

The term “oligonucleotide” refers to a short polymer of nucleotidesand/or nucleotide analogs. The term “RNA analog” refers to anpolynucleotide (e.g., a chemically synthesized polynucleotide) having atleast one altered or modified nucleotide as compared to a correspondingunaltered or unmodified RNA but retaining the same or similar nature orfunction as the corresponding unaltered or unmodified RNA. As discussedabove, the oligonucleotides may be linked with linkages which result ina lower rate of hydrolysis of the RNA analog as compared to an RNAmolecule with phosphodiester linkages. For example, the nucleotides ofthe analog may comprise methylenediol, ethylene diol, oxymethylthio,oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phosphoroamidate,and/or phosphorothioate linkages. In particular embodiments, RNAanalogues include sugar- and/or backbone-modified ribonucleotides and/ordeoxyribonucleotides. Such alterations or modifications can furtherinclude addition of non-nucleotide material, such as to the end(s) ofthe RNA or internally (at one or more nucleotides of the RNA). An RNAanalog need only be sufficiently similar to natural RNA that it has theability to mediate (mediates) RNA interference.

As used herein, exemplary oligonucleotides include, but are not limitedto, siRNAs, miRNAs, shRNAs, CRISPR guides, DNA oligonucleotides,antisense oligonucleotides, AAV oligonucleotides, gapmers, mixmers,miRNA inhibitors, SSOs, PMOs, PNAs and the like.

As used herein, the term “RNA interference” (“RNAi”) refers to aselective intracellular degradation of RNA. RNAi occurs in cellsnaturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAiproceeds via fragments cleaved from free dsRNA which direct thedegradative mechanism to other similar RNA sequences. Alternatively,RNAi can be initiated by the hand of man, for example, to silence theexpression of target genes.

As used herein, the term “hsiRNA” refers to an embodiment of thedouble-stranded RNAs provided herein, wherein the RNA molecule is fullychemically modified, including one or more hydrophobic modifications, asdescribed herein.

An RNAi agent, e.g., an RNA silencing agent, having a strand which is“sequence sufficiently complementary to a target mRNA sequence to directtarget-specific RNA interference (RNAi)” means that the strand has asequence sufficient to trigger the destruction of the target mRNA by theRNAi machinery or process.

As used herein, the term “isolated RNA” (e.g., “isolated siRNA” or“isolated siRNA precursor”) refers to RNA molecules which aresubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized.

As used herein, the term “RNA silencing” refers to a group ofsequence-specific regulatory mechanisms (e.g. RNA interference (RNAi),transcriptional gene silencing (TGS), post-transcriptional genesilencing (PTGS), quelling, co-suppression, translational repression andthe like) mediated by RNA molecules which result in the inhibition or“silencing” of the expression of a corresponding protein-coding gene.RNA silencing has been observed in many types of organisms, includingplants, animals, and fungi.

The term “discriminatory RNA silencing” refers to the ability of an RNAmolecule to substantially inhibit the expression of a “first” or“target” polynucleotide sequence while not substantially inhibiting theexpression of a “second” or “non-target” polynucleotide sequence, e.g.,when both polynucleotide sequences are present in the same cell. Incertain embodiments, the target polynucleotide sequence corresponds to atarget gene, while the non-target polynucleotide sequence corresponds toa non-target gene. In other embodiments, the target polynucleotidesequence corresponds to a target allele, while the non-targetpolynucleotide sequence corresponds to a non-target allele. In certainembodiments, the target polynucleotide sequence is the DNA sequenceencoding the regulatory region (e.g., promoter or enhancer elements) ofa target gene. In other embodiments, the target polynucleotide sequenceis a target mRNA encoded by a target gene.

A gene “involved” in a disease or disorder includes a gene, the normalor aberrant expression or function of which effects or causes thedisease or disorder or at least one symptom of said disease or disorder.

As used herein, the term “target gene” (e.g., the mutant allele of aheterozygous polymorphism, e.g., a heterozygous SNP) is a gene whoseexpression is to be substantially inhibited or “silenced.” Thissilencing can be achieved by RNA silencing, e.g., by cleaving the mRNAof the target gene or translational repression of the target gene. Theterm “non-target gene” (e.g., the wild-type allele) is a gene whoseexpression is not to be substantially silenced. In one embodiment, thepolynucleotide sequences of the target and non-target gene (e.g., mRNAencoded by the target and non-target genes) can differ by one or morenucleotides. In another embodiment, the target and non-target genes candiffer by one or more polymorphisms (e.g., single nucleotidepolymorphisms or SNPs). In another embodiment, the target and non-targetgenes can share less than 100% sequence identity. In another embodiment,the non-target gene may be a homolog (e.g., an ortholog or paralog) ofthe target gene.

A “target allele” is an allele (e.g., a SNP allele) whose expression isto be selectively inhibited or “silenced.” This silencing can beachieved by RNA silencing, e.g., by cleaving the mRNA of the target geneor target allele by a siRNA. The term “non-target allele” is an allele(e.g., the corresponding wild-type allele) whose expression is not to besubstantially silenced. In certain embodiments, the target andnon-target alleles can correspond to the same target gene. In otherembodiments, the target allele corresponds to, or is associated with, atarget gene, and the non-target allele corresponds to, or is associatedwith, a non-target gene. In one embodiment, the polynucleotide sequencesof the target and non-target alleles can differ by one or morenucleotides. In another embodiment, the target and non-target allelescan differ by one or more allelic polymorphisms (e.g., one or moreSNPs). In another embodiment, the target and non-target alleles canshare less than 100% sequence identity.

The term “polymorphism,” as used herein, refers to a variation (e.g.,one or more deletions, insertions, or substitutions) in a gene sequencethat is identified or detected when the same gene sequence fromdifferent sources or subjects (but from the same organism) are compared.For example, a polymorphism can be identified when the same genesequence from different subjects are compared. Identification of suchpolymorphisms is routine in the art, the methodologies being similar tothose used to detect, for example, breast cancer point mutations.Identification can be made, for example, from DNA extracted from asubject's lymphocytes, followed by amplification of polymorphic regionsusing specific primers to said polymorphic region. Alternatively, thepolymorphism can be identified when two alleles of the same gene arecompared.

In particular embodiments, the polymorphism is a single nucleotidepolymorphism (SNP). A variation in sequence between two alleles of thesame gene within an organism is referred to herein as an “allelicpolymorphism.” In certain embodiments, the allelic polymorphismcorresponds to a SNP allele. For example, the allelic polymorphism maycomprise a single nucleotide variation between the two alleles of a SNP,also referred to herein as a heterozygous SNP. The polymorphism can beat a nucleotide within a coding region but, due to the degeneracy of thegenetic code, no change in amino acid sequence is encoded.Alternatively, polymorphic sequences can encode a different amino acidat a particular position, but the change in the amino acid does notaffect protein function. Polymorphic regions can also be found innon-encoding regions of the gene. In particular embodiments, thepolymorphism is found in a coding region of the gene or in anuntranslated region (e.g., a 5′ UTR or 3′ UTR) of the gene.

As used herein, the term “allelic frequency” is a measure (e.g.,proportion or percentage) of the relative frequency of an allele (e.g.,a SNP allele) at a single locus in a population of individuals. Forexample, where a population of individuals carry n loci of a particularchromosomal locus (and the gene occupying the locus) in each of theirsomatic cells, then the allelic frequency of an allele is the fractionor percentage of loci that the allele occupies within the population. Inparticular embodiments, the allelic frequency of an allele (e.g. a SNPallele) is at least 10% (e.g., at least 15%, 20%, 25%, 30%, 35%, 40% ormore) in a sample population.

The term “gain-of-function mutation,” as used herein, refers to anymutation in a gene in which the protein encoded by said gene (i.e., themutant protein) acquires a function not normally associated with theprotein (i.e., the wild-type protein) causes or contributes to a diseaseor disorder. The gain-of-function mutation can be a deletion, addition,or substitution of a nucleotide or nucleotides in the gene which givesrise to the change in the function of the encoded protein. In oneembodiment, the gain-of-function mutation changes the function of themutant protein or causes interactions with other proteins. In anotherembodiment, the gain-of-function mutation causes a decrease in orremoval of normal wild-type protein, for example, by interaction of thealtered, mutant protein with said normal, wild-type protein.

As used herein, the term “gain-of-function disorder” refers to adisorder characterized by a gain-of-function mutation. In oneembodiment, the gain-of-function disorder is a neurodegenerative diseasecaused by a gain-of-function mutation, e.g., polyglutamine disordersand/or trinucleotide repeat diseases, for example, Huntington's disease.In another embodiment, the gain-of-function disorder is caused by again-of-function in an oncogene, the mutated gene product being again-of-function mutant, e.g., cancers caused by a mutation in the retoncogene (e.g., ret-1), for example, endocrine tumors, medullary thyroidtumors, parathyroid hormone tumors, multiple endocrine neoplasia type2,and the like. Additional exemplary gain-of-function disorders include,but are not limited to, Alzheimer's disease, amyotrophic lateralsclerosis (ALS), human immunodeficiency disorder (HIV), and slow channelcongenital myasthenic syndrome (SCCMS).

The term “trinucleotide repeat diseases,” as used herein, refers to anydisease or disorder characterized by an expanded trinucleotide repeatregion located within a gene, the expanded trinucleotide repeat regionbeing causative of the disease or disorder. Examples of trinucleotiderepeat diseases include, but are not limited to, spino-cerebellar ataxiatype 12 spino-cerebellar ataxia type 8, fragile X syndrome, fragile XEMental Retardation, Friedreich's ataxia and myotonic dystrophy.Preferred trinucleotide repeat diseases for treatment according to thepresent invention are those characterized or caused by an expandedtrinucleotide repeat region at the 5′ end of the coding region of agene, the gene encoding a mutant protein which causes or is causative ofthe disease or disorder. Certain trinucleotide diseases, for example,fragile X syndrome, where the mutation is not associated with a codingregion may not be suitable for treatment according to the methodologiesof the present invention, as there is no suitable mRNA to be targeted byRNAi. By contrast, disease such as Friedreich's ataxia may be suitablefor treatment according to the methodologies of the invention because,although the causative mutation is not within a coding region (i.e.,lies within an intron), the mutation may be within, for example, an mRNAprecursor (e.g., a pre-spliced mRNA precursor).

The term “polyglutamine disorder,” as used herein, refers to any diseaseor disorder characterized by an expanded of a (CAG)_(n) repeats at the5′ end of the coding region (thus encoding an expanded polyglutamineregion in the encoded protein). In one embodiment, polyglutaminedisorders are characterized by a progressive degeneration of nervecells. Examples of polyglutamine disorders include, but are not limitedto, Huntington's disease, spino-cerebellar ataxia type 1,spino-cerebellar ataxia type 2, spino-cerebellar ataxia type 3 (alsoknown as Machado-Joseph disease), and spino-cerebellar ataxia type 6,spino-cerebellar ataxia type 7, dentatoiubral-pallidoluysian atrophy andthe like.

The term “single nucleotide polymorphism disorder” or “SNP disorder”refers to a disorder characterized by a the presence of an SNP, e.g., aheterozygous SNP. SNP disorders include, but are not limited to,phenylketonuria, cystic fibrosis, sickle-cell anemia, albinism,Huntington's disease, myotonic dystrophy type 1, hypercholesterolemia(autosomal dominant, type B), neurofibromatosis (type 1), polycystickidney disease (1 and 2), hemophilia A, Duchenne's muscular dystrophy,X-linked hypophosphatemic rickets, Rett's syndrome, non-obstructivespermatogenic failure and the like. An exemplary heterozygous SNPdisorder is Huntington's disease.

In certain aspects, a double-stranded RNA (dsRNA) is provided comprisinga first strand of about 15-35 nucleotides that is complementary to aregion of a gene comprising an allelic polymorphism, and a second strandof about 15-35 nucleotides that is complementary to at least a portionof the first strand, wherein the first strand comprises a singlenucleotide polymorphism (SNP) position nucleotide at a position 2 to 7from the 5′ end that is complementary to the allelic polymorphism; and amismatch (MM) position nucleotide located 2-11 nucleotides from the SNPposition nucleotide that is a mismatch with a nucleotide in the gene. Insome cases, the MM position is located 2 to 10 nucleotides from the SNPposition. In some cases, the SNP position nucleotide is any one ofpositions 2 to 6 from the 5′ end. In exemplary embodiments, the SNPposition nucleotide is at a position 2, 4 or 6 from the 5′ end and themismatch (MM) position nucleotide is located 2-6 nucleotides from theSNP position nucleotide. In some cases, the SNP position nucleotide isany one of positions 2 to 6 from the 5′ end and the nucleotide islocated 2-6 nucleotides from the SNP position nucleotide.

As used herein, a “single nucleotide polymorphism position nucleotide”or a “SNP position nucleotide” refers to the position of an RNAdescribed herein (e.g., the first strand of a dsRNA) that corresponds tothe polymorphic position of a target nucleic acid sequence (i.e., eitherthe mutant nucleotide corresponding to the SNP allele or the wild-typenucleotide corresponding to the wild-type allele). For example, a strandmay be labeled “SNP2,” “SNP3,” or “SNP3” to denote the position of theSNP as being 2, 3, or 4 nucleotides from the 5′ end of the strand.

In certain exemplary embodiments, a SNP position nucleotide is within aseed region. In certain exemplary embodiments, a SNP position nucleotideis located from position 2 to position 7 from the 5′ end, from position2 to position 6 from the 5′ end, or from position 2 to position 5 fromthe 5′ end. In certain exemplary embodiments, a SNP position nucleotideis located at position 2 from the 5′ end, at position 3 from the 5′ end,at position 4 from the 5′ end, at position from the 5′ end, at position6 from the 5′ end, or at position 7 from the 5′ end of an RNA describedherein (e.g., the first strand of a dsRNA). In certain exemplaryembodiments, a SNP position nucleotide is located at a position setforth in Tables 5-7.

As used herein, the term “seed region” refers to a six-nucleotidestretch corresponding to positions 2-7 from the 5′ end of an RNA strand.siRNA recognition of the target mRNA is believed to be conferred by theseed region of its antisense strand.

As used herein, a “mismatch position nucleotide” or a “MM positionnucleotide” refers to the position of an RNA described herein (e.g., thefirst strand of a dsRNA) that is in a position that does not correspondto the SNP position nucleotide. A MM position nucleotide can be definedby its position from the 5′ end or the 3′ end of an RNA described herein(e.g., the 5′ or the 3′ end of first strand of a dsRNA), or defined byits position relative to a SNP position nucleotide of an RNA describedherein (e.g., a first strand of a dsRNA).

In certain exemplary embodiments, a MM position nucleotide is located2-11 nucleotides, 2-10 nucleotides, 2-9 nucleotides, 2-8 nucleotides,2-7 nucleotides, or 2-6 nucleotides from a SNP position nucleotide. Incertain exemplary embodiments, a MM position nucleotide is located 11nucleotides, 10 nucleotides, 9 nucleotides, 8 nucleotides, 7nucleotides, 6 nucleotides, 5 nucleotides, 4 nucleotides, 3 nucleotidesor 2 nucleotides from a SNP position nucleotide. In certain exemplaryembodiments, a MM position nucleotide is located at a position set forthin Tables 5-7.

In one embodiment, an RNA described herein (e.g., the first strand of adsRNA) is homologous to an allelic polymorphism except for onemismatched oligonucleotide at a particular position relative to thenucleotide corresponding to the allelic polymorphism. In certainembodiments, the mismatch is within about 6 nucleotides of the SNPposition nucleotide, within about 5 nucleotides of the SNP positionnucleotide, within about 4 nucleotides of the SNP position nucleotide,within about 3 nucleotide of the SNP position nucleotide, within about 2nucleotide of the SNP position nucleotide, or within about 1 nucleotideof the SNP position nucleotide. In particular embodiments, the mismatchis not adjacent to a SNP position nucleotide.

In another embodiment, a SNP position nucleotide is at position 2, 3, 4,5, or 6 from the 5′ end. In an embodiment, a SNP position nucleotide isat position 2 from the 5′ end. In an embodiment, is at position 3 fromthe 5′ end. In an embodiment, a SNP position nucleotide is at position 4from the 5′ end. In an embodiment, a SNP position nucleotide is atposition 5 from the 5′ end. In an embodiment, a SNP position nucleotideis at position 6 from the 5′ end.

In certain exemplary embodiments, an RNA described herein (e.g., thefirst strand of a dsRNA) comprises a MM position nucleotide at position5, 7, 8, 11, 14, 15 or 16 from the 5′ end. In certain exemplaryembodiments, an RNA described herein (e.g., the first strand of a dsRNA)comprises a MM position nucleotide 1, 2, 3, 4, 5, 8, 9, 10 or 11nucleotides from the SNP position nucleotide.

In certain exemplary embodiments, an RNA described herein (e.g., thefirst strand of a dsRNA) comprises a SNP position nucleotide (referencedfrom the 5′ end)—MM position nucleotide (referenced from the 5′ end)combination selected from the group consisting of 2-7, 4-7, 4-8, 4-15,6-5, 6-8, 6-11, 6-14, 6-16, 3-5, 3-7 and 3-8.

In a particularly exemplary embodiment, an RNA described herein (e.g.,the first strand of a dsRNA) comprises an SNP position nucleotide atposition 6 from the 5′ end and a MM position nucleotide at position 11from the 5′ end. In another particularly exemplary embodiment, an RNAdescribed herein (e.g., the first strand of a dsRNA) comprises an SNPposition nucleotide at position 4 from the 5′ end and a mismatch atposition 7 from the 5′ end.

In one aspect, the double-stranded RNAs provided herein selectivelysilence a mutant allele having an allelic polymorphism. In anembodiment, the double-stranded RNAs provided herein silence a mutantallele having an allelic polymorphism and do not affect the wild-typeallele of the same gene. In another embodiment, the double-stranded RNAsprovided herein silence a mutant allele having an allelic polymorphismand silence the wild-type allele of the same gene to a lesser extentthan the mutant allele.

Accordingly, in one aspect, the present invention provides a method oftreating a subject having or at risk of having a disease characterizedor caused by a mutant protein associated with an allelic polymorphism byadministering to the subject an effective amount of an RNAi agenttargeting an allelic polymorphism within a gene encoding a mutantprotein (e.g., huntingtin protein), such that sequence-specificinterference of a gene occurs resulting in an effective treatment forthe disease.

In one aspect, RNA silencing agents disclosed herein preferentiallysilence a mutant allele comprising a polymorphism more efficiently thanthe corresponding wild-type allele. In certain exemplary embodiments,dsRNAs disclosed herein silence the allele comprising a polymorphismabout 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about80%, or about 90% more than the corresponding wild-type allele. In anembodiment, RNA silencing agents disclosed herein silence the allelecomprising a polymorphism at least about 50% more than the correspondingwild-type allele. In certain exemplary embodiments, dsRNAs disclosedherein silence the allele comprising a polymorphism at least about 5times, about 10 times, about 15 times, about 20 times, about 25 times,about 30 times, about 35 times, about 40 times, about 45 times, about 50times, about 55 times, about 60 times, about 65 times, about 70 times,about 75 times, about 80 times, about 85 times, about 90 times, about 95times, about 100 times, about 110 times, about 120 times, about 130times, about 140 times, about 150 times, about 160 times, about 170times, about 180 times, about 190 times, about 200 times, about 250times, about 300 times, about 350 times, about 400 times, about 450times, or up to about 500 times the level of silencing of thecorresponding wild-type allele.

As used herein, the term “antisense strand” of an RNA silencing agent,e.g., an siRNA or RNA silencing agent, refers to a strand that issubstantially complementary to a section of about 10-50 nucleotides,e.g., about 15-30, 16-25, 18-23 or 19-22 nucleotides of the mRNA of thegene targeted for silencing. The antisense strand or first strand hassequence sufficiently complementary to the desired target mRNA sequenceto direct target-specific silencing, e.g., complementarity sufficient totrigger the destruction of the desired target mRNA by the RNAi machineryor process (RNAi interference) or complementarity sufficient to triggertranslational repression of the desired target mRNA.

The term “sense strand” or “second strand” of an RNA silencing agent,e.g., an siRNA or RNA silencing agent, refers to a strand that iscomplementary to the antisense strand or first strand. Antisense andsense strands can also be referred to as first or second strands, thefirst or second strand having complementarity to the target sequence andthe respective second or first strand having complementarity to saidfirst or second strand. miRNA duplex intermediates or siRNA-likeduplexes include a miRNA strand having sufficient complementarity to asection of about 10-50 nucleotides of the mRNA of the gene targeted forsilencing and a miRNA* strand having sufficient complementarity to forma duplex with the miRNA strand.

As used herein, the term “antisense oligonucleotide” or “ASO” refers toa nucleic acid (e.g., an RNA), having sufficient sequencecomplementarity to a target an RNA (e.g., a SNP-containing mRNA or aSNP-containing pre-mRNA) in order to block a region of a target RNA inan effective manner, e.g., in a manner effective to inhibit translationof a target mRNA and/or splicing of a target pre-mRNA. An antisenseoligonucleotide having a “sequence sufficiently complementary to atarget RNA” means that the antisense agent has a sequence sufficient tomask a binding site for a protein that would otherwise modulate splicingand/or that the antisense agent has a sequence sufficient to mask abinding site for a ribosome and/or that the antisense agent has asequence sufficient to alter the three-dimensional structure of thetargeted RNA to prevent splicing and/or translation.

As used herein, the “5′ end,” as in the 5′ end of an antisense strand,refers to the 5′ terminal nucleotides, e.g., between one and about 5nucleotides at the 5′ terminus of the antisense strand. As used herein,the “3′ end,” as in the 3′ end of a sense strand, refers to the region,e.g., a region of between one and about 5 nucleotides, that iscomplementary to the nucleotides of the 5′ end of the complementaryantisense strand.

As used herein, the term “base pair” refers to the interaction betweenpairs of nucleotides (or nucleotide analogs) on opposing strands of anoligonucleotide duplex (e.g., a duplex formed by a strand of a RNAsilencing agent and a target mRNA sequence), due primarily to H-bonding,Van der Waals interactions, and the like between said nucleotides (ornucleotide analogs). As used herein, the term “bond strength” or “basepair strength” refers to the strength of the base pair.

As used herein, the term “mismatched base pair” refers to a base pairconsisting of non-complementary or non-Watson-Crick base pairs, forexample, not normal complementary G:C, A:T or A:U base pairs. As usedherein the term “ambiguous base pair” (also known as anon-discriminatory base pair) refers to a base pair formed by auniversal nucleotide.

Linkers useful in conjugated compounds of the invention include glycolchains (e.g., polyethylene glycol), alkyl chains, peptides, RNA, DNA,and combinations thereof. As used herein, the abbreviation “TEG” refersto triethylene glycol.

Design of Oligonucleotides

In certain embodiments, an oligonucleotide, e.g., an siRNA, of theinvention is a duplex consisting of a sense strand and complementaryantisense strand, the antisense strand having sufficient complementaryto an target mRNA containing an allelic polymorphism to mediate RNAi. Inexemplary embodiments, the siRNA molecule has a length from about 10-50or more nucleotides, i.e., each strand comprises 10-50 nucleotides (ornucleotide analogs). In particularly exemplary embodiments, the siRNAmolecule has a length from about 15-35, e.g., about 15, about 16, about17, about 18, about 19, about 20, about 21, about 22, about 23, about24, about 25, about 26, about 27, about 28, about 29, about 30, about31, about 32, about 33, about 34 or about 35 nucleotides in each strand,wherein one of the strands is sufficiently complementary to a targetregion. In exemplary embodiments, the strands are aligned such thatthere are at least 1, 2, or 3 bases at the end of the strands which donot align (i.e., for which no complementary bases occur in the opposingstrand) such that an overhang of 1, 2 or 3 residues occurs at one orboth ends of the duplex when strands are annealed. In exemplaryembodiments, the siRNA molecule has a length from about 10-50 or morenucleotides, i.e., each strand comprises 10-50 nucleotides (ornucleotide analogs). In particularly exemplary embodiments, the siRNAmolecule has a length from about 15-35, e.g., about 15, about 16, about17, about 18, about 19, about 20, about 21, about 22, about 23, about24, about 25, about 26, about 27, about 28, about 29, about 30, about31, about 32, about 33, about 34 or about 35 nucleotides in each strand,wherein one of the strands is substantially complementary to a targetsequence containing an allelic polymorphism, and the other strand iscomplementary or substantially complementary to the first strand. In anembodiment, the siRNA molecule in fully complementary to a targetsequence containing an allelic polymorphism except for one additionalmismatch, also known as secondary mismatch.

Generally, siRNAs can be designed by using any method known in the art,for instance, by using the following protocol:

1. The siRNA may be specific for a target sequence which contains anallelic polymorphism. In exemplary embodiments, the first strand issubstantially complementary to the target sequence containing an allelicpolymorphism having one mismatch to the target sequence containing anallelic polymorphism, and the other strand is substantiallycomplementary to the first strand. In an embodiment, the target sequenceis outside a coding region of the target gene. Exemplary targetsequences are selected from the 5′ untranslated region (5′-UTR) or anintronic region of a target gene. Cleavage of mRNA at these sites shouldeliminate translation of corresponding mutant protein. Target sequencesfrom other regions of the htt gene are also suitable for targeting. Asense strand is designed based on the target sequence. Further, siRNAswith lower G/C content (35-55%) may be more active than those with G/Ccontent higher than 55%. Thus in one embodiment, the invention includesnucleic acid molecules having 35-55% G/C content.

2. The sense strand of the siRNA is designed based on the sequence ofthe selected target site and the position of the allelic polymorphism.In exemplary embodiments, the RNA silencing agents of the invention donot elicit a PKR response (i.e., are of a sufficiently short length).However, longer RNA silencing agents may be useful, for example, in celltypes incapable of generating a PKR response or in situations where thePKR response has been down-regulated or dampened by alternative means.

The siRNA molecules of the invention have sufficient complementaritywith the target sequence such that the siRNA can mediate RNAi. Ingeneral, siRNA containing nucleotide sequences sufficiently identical toa target sequence portion of the target gene to effect RISC-mediatedcleavage of the target gene are used. Accordingly, in an exemplaryembodiment, the sense strand of the siRNA is designed have to have asequence sufficiently identical to a portion of the target whichcontains an allelic polymorphism. The invention has the advantage ofbeing able to tolerate certain sequence variations to enhance efficiencyand specificity of RNAi. In an aspect, the sense strand has 1 mismatchednucleotide with a target region containing an allelic polymorphism, suchas a target region that differs by at least one base pair between awild-type and mutant allele, e.g., a target region comprising thegain-of-function mutation, and the other strand is identical orsubstantially identical to the first strand. In some embodiments, themismatch is 4 nucleotides upstream, 3 nucleotides upstream nucleotidecorresponding to the allelic polymorphism, 2 nucleotides upstreamnucleotide corresponding to the allelic polymorphism, 1 nucleotideupstream, 1 nucleotide downstream nucleotide corresponding to theallelic polymorphism, 2 nucleotides downstream nucleotide correspondingto the allelic polymorphism, 3 nucleotides downstream nucleotidecorresponding to the allelic polymorphism, 4 nucleotides downstreamnucleotide corresponding to the allelic polymorphism, or 5 nucleotidesdownstream nucleotide corresponding to the allelic polymorphism. Incertain embodiments, the mismatch is not adjacent to the nucleotidecorresponding to the allelic polymorphism. Moreover, siRNA sequenceswith small insertions or deletions of 1 or 2 nucleotides may also beeffective for mediating RNAi. Alternatively, siRNA sequences withnucleotide analog substitutions or insertions can be effective forinhibition.

Sequence identity may be determined by sequence comparison and alignmentalgorithms known in the art. To determine the percent identity of twonucleic acid sequences (or of two amino acid sequences), the sequencesare aligned for optimal comparison purposes (e.g., gaps can beintroduced in the first sequence or second sequence for optimalalignment). The nucleotides (or amino acid residues) at correspondingnucleotide (or amino acid) positions are then compared. When a positionin the first sequence is occupied by the same residue as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., percent (%) homology=number of identicalpositions/total number of positions ×100), optionally penalizing thescore for the number of gaps introduced and/or length of gapsintroduced.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In one embodiment, the alignment generated over a certainportion of the sequence aligned having sufficient identity but not overportions having low degree of identity (i.e., a local alignment). Anexemplary, non-limiting example of a local alignment algorithm utilizedfor the comparison of sequences is the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithmis incorporated into the BLAST programs (version 2.0) of Altschul, etal. (1990) J. Mol. Biol. 215:403-10.

In another embodiment, the alignment is optimized by introducingappropriate gaps and percent identity is determined over the length ofthe aligned sequences (i.e., a gapped alignment). To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. In another embodiment, the alignment is optimized byintroducing appropriate gaps and percent identity is determined over theentire length of the sequences aligned (i.e., a global alignment). Anexemplary, non-limiting example of a mathematical algorithm utilized forthe global comparison of sequences is the algorithm of Myers and Miller,CABIOS (1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

3. The antisense or guide strand of the siRNA is routinely the samelength as the sense strand and includes complementary nucleotides. Inone embodiment, the guide and sense strands are fully complementary,i.e., the strands are blunt-ended when aligned or annealed. In anotherembodiment, the strands of the siRNA can be paired in such a way as tohave a 3′ overhang of 1 to 4, e.g., 2, nucleotides. Overhangs cancomprise (or consist of) nucleotides corresponding to the target genesequence (or complement thereof). Alternatively, overhangs can comprise(or consist of) deoxyribonucleotides, for example dTs, or nucleotideanalogs, or other suitable non-nucleotide material. Thus in anotherembodiment, the nucleic acid molecules may have a 3′ overhang of 2nucleotides, such as TT. The overhanging nucleotides may be either RNAor DNA. As noted above, it is desirable to choose a target regionwherein the mutant:wild-type mismatch is a purine:purine mismatch.

4. Using any method known in the art, compare the potential targets tothe appropriate genome database (human, mouse, rat, etc.) and eliminatefrom consideration any target sequences with significant homology toother coding sequences. One such method for such sequence homologysearches is known as BLAST, which is available at National Center forBiotechnology Information website.

5. Select one or more sequences that meet the criteria for evaluation.

Further general information about the design and use of siRNA may befound in “The siRNA User Guide,” available at The Max-Plank-Institut furBiophysikalishe Chemie website.

Alternatively, the siRNA may be defined functionally as a nucleotidesequence (or oligonucleotide sequence) that is capable of hybridizingwith the target sequence (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. hybridization for 12-16 hours; followed bywashing). Additional exemplary hybridization conditions includehybridization at 70° C. in 1×SSC or 50° C. in 1×SSC, 50% formamidefollowed by washing at 70° C. in 0.3×SSC or hybridization at 70° C. in4×SSC or 50° C. in 4×SSC, 50% formamide followed by washing at 67° C. in1×SSC. The hybridization temperature for hybrids anticipated to be lessthan 50 base pairs in length should be 5-10° C. less than the meltingtemperature (Tm) of the hybrid, where Tm is determined according to thefollowing equations. For hybrids less than 18 base pairs in length, Tm(°C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49base pairs in length, Tm(° C.)=81.5+16.6(log 10[Na+])+0.41(%G+C)−(600/N), where N is the number of bases in the hybrid, and [Na+] isthe concentration of sodium ions in the hybridization buffer ([Na+] for1×SSC=0.165 M). Additional examples of stringency conditions forpolynucleotide hybridization are provided in Sambrook, J., E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters9 and 11, and Current Protocols in Molecular Biology, 1995, F. M.Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and6.3-6.4, incorporated herein by reference in its entirety for allpurposes.

Negative control siRNAs should have the same nucleotide composition asthe selected siRNA, but without significant sequence complementarity tothe appropriate genome. Such negative controls may be designed byrandomly scrambling the nucleotide sequence of the selected siRNA. Ahomology search can be performed to ensure that the negative controllacks homology to any other gene in the appropriate genome. In addition,negative control siRNAs can be designed by introducing one or more basemismatches into the sequence.

6. To validate the effectiveness by which siRNAs destroy target mRNAs(e.g., wild-type or mutant huntingtin mRNA), the siRNA may be incubatedwith target cDNA (e.g., huntingtin cDNA) in a Drosophila-based in vitromRNA expression system. Radiolabeled with ³²P, newly synthesized targetmRNAs (e.g., huntingtin mRNA) are detected autoradiographically on anagarose gel. The presence of cleaved target mRNA indicates mRNA nucleaseactivity. Suitable controls include omission of siRNA and use ofnon-target cDNA. Alternatively, control siRNAs are selected having thesame nucleotide composition as the selected siRNA, but withoutsignificant sequence complementarity to the appropriate target gene.Such negative controls can be designed by randomly scrambling thenucleotide sequence of the selected siRNA. A homology search can beperformed to ensure that the negative control lacks homology to anyother gene in the appropriate genome. In addition, negative controlsiRNAs can be designed by introducing one or more base mismatches intothe sequence.

siRNAs may be designed to target any of the target sequences describedsupra. Said siRNAs comprise an antisense strand which is sufficientlycomplementary with the target sequence to mediate silencing of thetarget sequence. In certain embodiments, the RNA silencing agent is asiRNA.

Sites of siRNA-mRNA complementation are selected, which result inoptimal mRNA specificity and maximal mRNA cleavage.

siRNA-Like Molecules

siRNA-like molecules of the invention have a sequence (i.e., have astrand having a sequence) that is “sufficiently complementary” to atarget sequence of an mRNA (e.g. an htt mRNA) to direct gene silencingeither by RNAi or translational repression. siRNA-like molecules aredesigned in the same way as siRNA molecules, but the degree of sequenceidentity between the sense strand and target RNA approximates thatobserved between an miRNA and its target. In general, as the degree ofsequence identity between a miRNA sequence and the corresponding targetgene sequence is decreased, the tendency to mediate post-transcriptionalgene silencing by translational repression rather than RNAi isincreased. Therefore, in an alternative embodiment, wherepost-transcriptional gene silencing by translational repression of thetarget gene is desired, the miRNA sequence has partial complementaritywith the target gene sequence. In certain embodiments, the miRNAsequence has partial complementarity with one or more short sequences(complementarity sites) dispersed within the target mRNA (e.g., withinthe 3′-UTR of the target mRNA) (Hutvagner and Zamore, Science, 2002;Zeng et al., Mol. Cell, 2002; Zeng et al., RNA, 2003; Doench et al.,Genes & Dev., 2003). Since the mechanism of translational repression iscooperative, multiple complementarity sites (e.g., 2, 3, 4, 5, or 6) maybe targeted in certain embodiments.

The capacity of a siRNA-like duplex to mediate RNAi or translationalrepression may be predicted by the distribution of non-identicalnucleotides between the target gene sequence and the nucleotide sequenceof the silencing agent at the site of complementarity. In oneembodiment, where gene silencing by translational repression is desired,at least one non-identical nucleotide is present in the central portionof the complementarity site so that duplex formed by the miRNA guidestrand and the target mRNA contains a central “bulge” (Doench J G etal., Genes & Dev., 2003). In another embodiment 2, 3, 4, 5, or 6contiguous or non-contiguous non-identical nucleotides are introduced.The non-identical nucleotide may be selected such that it forms a wobblebase pair (e.g., G:U) or a mismatched base pair (G:A, C:A, C:U, G:G,A:A, C:C, U:U). In a further exemplary embodiment, the “bulge” iscentered at nucleotide positions 12 and 13 from the 5′ end of the miRNAmolecule.

Modified RNA Silencing Agents

In certain aspects of the invention, an RNA silencing agent (or anyportion thereof) of the invention as described supra may be modifiedsuch that the activity of the agent is further improved. For example,the RNA silencing agents described in above may be modified with any ofthe modifications described infra. The modifications can, in part, serveto further enhance target discrimination, to enhance stability of theagent (e.g., to prevent degradation), to promote cellular uptake, toenhance the target efficiency, to improve efficacy in binding (e.g., tothe targets), to improve patient tolerance to the agent, and/or toreduce toxicity.

1) Modifications to Enhance Target Discrimination

In certain embodiments, the RNA silencing agents of the invention may besubstituted with a destabilizing nucleotide to enhance single nucleotidetarget discrimination (see U.S. application Ser. No. 11/698,689, filedJan. 25, 2007 and U.S. Provisional Application No. 60/762,225 filed Jan.25, 2006, both of which are incorporated herein by reference). Such amodification may be sufficient to abolish the specificity of the RNAsilencing agent for a non-target mRNA (e.g. wild-type mRNA), withoutappreciably affecting the specificity of the RNA silencing agent for atarget mRNA (e.g. gain-of-function mutant mRNA).

In certain exemplary embodiments, the RNA silencing agents of theinvention are modified by the introduction of at least one universalnucleotide in the antisense strand thereof. Universal nucleotidescomprise base portions that are capable of base pairing indiscriminatelywith any of the four conventional nucleotide bases (e.g. A, G, C, U). Auniversal nucleotide is typically utilized because it has relativelyminor effect on the stability of the RNA duplex or the duplex formed bythe guide strand of the RNA silencing agent and the target mRNA.Exemplary universal nucleotide include those having an inosine baseportion or an inosine analog base portion selected from the groupconsisting of deoxyinosine (e.g. 2′-deoxyinosine),7-deaza-2′-deoxyinosine, 2′-aza-2′-deoxyinosine, PNA-inosine,morpholino-inosine, LNA-inosine, phosphoramidate-inosine,2′-O-methoxyethyl-inosine, and 2′-OMe-inosine. In particularly exemplaryembodiments, the universal nucleotide is an inosine residue or anaturally occurring analog thereof.

In certain embodiments, the RNA silencing agents of the invention aremodified by the introduction of at least one destabilizing nucleotidewithin 11 nucleotides from a specificity-determining nucleotide (e.g.,within 11 nucleotides from the nucleotide which recognizes thedisease-related polymorphism (e.g., a SNP position nucleotide)). Forexample, the destabilizing nucleotide may be introduced at a positionthat is within 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide(s) from aspecificity-determining nucleotide. In exemplary embodiments, thedestabilizing nucleotide is introduced at a position which is 3nucleotides from the specificity-determining nucleotide (i.e., such thatthere are 2 stabilizing nucleotides between the destabilizing nucleotideand the specificity-determining nucleotide). In RNA silencing agentshaving two strands or strand portions (e.g., siRNAs and shRNAs), thedestabilizing nucleotide may be introduced in the strand or strandportion that does not contain the specificity-determining nucleotide. Inparticular exemplary embodiments, the destabilizing nucleotide isintroduced in the same strand or strand portion that contains thespecificity-determining nucleotide.

In certain embodiments, the RNA silencing agents of the invention aremodified by the introduction of a modified intersubunit linkage ofFormula 1:

wherein:

D is selected from the group consisting of O, OCH₂, OCH, CH₂, and CH;

C is selected from the group consisting of O⁻, OH, OR¹, NH⁻, NH₂, S⁻,and SH;

A is selected from the group consisting of O and CH₂;

R¹ is a protecting group;

is an optional double bond; and

the intersubunit is bridging two optionally modified nucleosides.

In an embodiment, when C is O⁻, either A or D is not O.

In an embodiment, D is CH2. In another embodiment, the modifiedintersubunit linkage of Formula VIII is a modified intersubunit linkageof Formula 2:

In an embodiment, D is O. In another embodiment, the modifiedintersubunit linkage of Formula VIII is a modified intersubunit linkageof Formula 3:

In an embodiment, D is CH₂. In another embodiment, the modifiedintersubunit linkage of Formula VIII is a modified intersubunit linkageof Formula 4:

In another embodiment, the modified intersubunit linkage is a modifiedintersubunit linkage of Formula 5:

In an embodiment, D is OCH₂. In another embodiment, the modifiedintersubunit linkage is a modified intersubunit linkage of Formula 6:

In another embodiment, the modified intersubunit linkage of Formula VIIis a modified intersubunit linkage of Formula 7:

In certain embodiments, the RNA silencing agents of the invention aremodified by the introduction of one or more of the intersubunit linkersof FIG. 43 . In an exemplary embodiment, an intersubunit linker of FIG.43 is inserted between the SNP position nucleotide and a nucleotide at aposition directly adjacent to and on either side of the SNP positionnucleotide of the antisense strand.

In certain embodiments, the RNA silencing agents of the invention aremodified by the introduction of one or more vinyl phosphonate (VP)motifs in the intersubunit linker having the following formula:

In certain embodiments, a VP motif is inserted at any position(s) of anoligonucleotide, e.g., an RNA. For example, for an oligonucleotidehaving a length of 20 nucleotides, a VP motif can be inserted atposition 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12,12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19 or 19-20 and at anycombinations of these.

In certain exemplary embodiments, a VP motif is inserted at one or moreof positions 1-2, 5-6, 6-7, 10-11, 18-19 and/or 19-20 of the antisensestrand.

In other exemplary embodiments, a VP motif is inserted at one or more ofpositions 1-2, 6-7, 10-11 and/or 19-20 of the antisense strand.

In an exemplary embodiment, a VP motif is inserted next to (i.e.,between a SNP position nucleotide and a nucleotide at a positiondirectly adjacent to and on either side of) the SNP position nucleotideof the antisense strand. In another exemplary embodiment, a VP motif isinserted next to (i.e., between a MM position nucleotide and anucleotide at a position directly adjacent to and on either side of) theMM position nucleotide of the antisense strand.

2) Modifications to Enhance Efficacy and Specificity

In certain embodiments, the RNA silencing agents of the invention may bealtered to facilitate enhanced efficacy and specificity in mediatingRNAi according to asymmetry design rules (see U.S. Pat. Nos. 8,309,704,7,750,144, 8,304,530, 8,329,892 and 8,309,705). Such alterationsfacilitate entry of the antisense strand of the siRNA (e.g., a siRNAdesigned using the methods of the invention or an siRNA produced from ashRNA) into RISC in favor of the sense strand, such that the antisensestrand preferentially guides cleavage or translational repression of atarget mRNA, and thus increasing or improving the efficiency of targetcleavage and silencing. In exemplary embodiments, the asymmetry of anRNA silencing agent is enhanced by lessening the base pair strengthbetween the antisense strand 5′ end (AS 5′) and the sense strand 3′ end(S 3′) of the RNA silencing agent relative to the bond strength or basepair strength between the antisense strand 3′ end (AS 3′) and the sensestrand 5′ end (S ′5) of said RNA silencing agent.

In one embodiment, the asymmetry of an RNA silencing agent of theinvention may be enhanced such that there are fewer G:C base pairsbetween the 5′ end of the first or antisense strand and the 3′ end ofthe sense strand portion than between the 3′ end of the first orantisense strand and the 5′ end of the sense strand portion. In anotherembodiment, the asymmetry of an RNA silencing agent of the invention maybe enhanced such that there is at least one mismatched base pair betweenthe 5′ end of the first or antisense strand and the 3′ end of the sensestrand portion. In an exemplary embodiment, the mismatched base pair isselected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C andU:U. In another embodiment, the asymmetry of an RNA silencing agent ofthe invention may be enhanced such that there is at least one wobblebase pair, e.g., G:U, between the 5′ end of the first or antisensestrand and the 3′ end of the sense strand portion. In anotherembodiment, the asymmetry of an RNA silencing agent of the invention maybe enhanced such that there is at least one base pair comprising a rarenucleotide, e.g., inosine (I). In exemplary embodiments, the base pairis selected from the group consisting of an I:A, I:U and I:C. In yetanother embodiment, the asymmetry of an RNA silencing agent of theinvention may be enhanced such that there is at least one base paircomprising a modified nucleotide. In particular embodiments, themodified nucleotide is selected from the group consisting of 2-amino-G,2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

In certain embodiments, the RNA silencing agents of the invention arealtered at one or more intersubunit linkages in an oligonucleotide bythe introduction of a vinyl phosphonate (VP) motif having the followingformula:

3) RNA Silencing Agents with Enhanced Stability

The RNA silencing agents of the present invention can be modified toimprove stability in serum or in growth medium for cell cultures. Inorder to enhance the stability, the 3′-residues may be stabilizedagainst degradation, e.g., they may be selected such that they consistof purine nucleotides, particularly adenosine or guanosine nucleotides.Alternatively, substitution of pyrimidine nucleotides by modifiedanalogues, e.g., substitution of uridine by 2′-deoxythymidine istolerated and does not affect the efficiency of RNA interference.

In a particular aspect, the invention features RNA silencing agents thatinclude first and second strands wherein the second strand and/or firststrand is modified by the substitution of internal nucleotides withmodified nucleotides, such that in vivo stability is enhanced ascompared to a corresponding unmodified RNA silencing agent. As definedherein, an “internal” nucleotide is one occurring at any position otherthan the 5′ end or 3′ end of nucleic acid molecule, polynucleotide oroligonucleotide. An internal nucleotide can be within a single-strandedmolecule or within a strand of a duplex or double-stranded molecule. Inone embodiment, the sense strand and/or antisense strand is modified bythe substitution of at least one internal nucleotide. In anotherembodiment, the sense strand and/or antisense strand is modified by thesubstitution of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or more internal nucleotides. Inanother embodiment, the sense strand and/or antisense strand is modifiedby the substitution of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of theinternal nucleotides. In yet another embodiment, the sense strand and/orantisense strand is modified by the substitution of all of the internalnucleotides.

In a particular embodiment of the present invention, the RNA silencingagents may contain at least one modified nucleotide analogue. Thenucleotide analogues may be located at positions where thetarget-specific silencing activity, e.g., the RNAi mediating activity ortranslational repression activity is not substantially effected, e.g.,in a region at the 5′-end and/or the 3′-end of the siRNA molecule.Particularly, the ends may be stabilized by incorporating modifiednucleotide analogues.

In certain embodiments, the RNA silencing agents of the invention arealtered at one or more intersubunit linkages in an oligonucleotide bythe introduction of a vinyl phosphonate (VP) motif having the followingformula:

A variety of oligonucleotide types (e.g., gapmers, mixmers, miRNAinhibitors, splice-switching oligonucleotides (“SSOs”),phosphorodiamidate morpholino oligonucleotides (“PMOs”), peptide nucleicacids (“PNAs”) and the like) can be used in the oligonucleotidesdescribed herein, optionally utilizing various combinations ofmodifications (e.g., chemical modifications) and/or conjugationsdescribed herein and in, e.g., U.S. Ser. No. 15/089,423; U.S. Ser. No.15/236,051; U.S. Ser. No. 15/419,593; U.S. Ser. No. 15/697,120 and U.S.Pat. No. 9,809,817; and U.S. Ser. No. 15/814,350 and U.S. Pat. No.9,862,350, each of which is incorporated herein by reference in itsentirety for all purposes.

Exemplary nucleotide analogues include sugar- and/or backbone-modifiedribonucleotides (i.e., include modifications to the phosphate-sugarbackbone). For example, the phosphodiester linkages of natural RNA maybe modified to include at least one of a nitrogen or sulfur heteroatom.In exemplary backbone-modified ribonucleotides, the phosphoester groupconnecting to adjacent ribonucleotides is replaced by a modified group,e.g., of phosphothioate group. In exemplary sugar-modifiedribonucleotides, the 2′ OH-group is replaced by a group selected from H,OR, R, halo, SH, SR, NH₂, NHR, NR₂ or ON, wherein R is C₁-C₆ alkyl,alkenyl or alkynyl and halo is F, Cl, Br or I.

In particular embodiments, the modifications are 2′-fluoro, 2′-aminoand/or 2′-thio modifications. Particularly exemplary modificationsinclude 2′-fluoro-cytidine, 2′-fluoro-uridine, 2′-fluoro-adenosine,2′-fluoro-guanosine, 2′-amino-cytidine, 2′-amino-uridine,2′-amino-adenosine, 2′-amino-guanosine, 2,6-diaminopurine,4-thio-uridine, and/or 5-amino-allyl-uridine. In a particularembodiment, the 2′-fluoro ribonucleotides are every uridine andcytidine. Additional exemplary modifications include 5-bromo-uridine,5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine,2′-amino-butyryl-pyrene-uridine, 5-fluoro-cytidine, and5-fluoro-uridine. 2′-deoxy-nucleotides and 2′-Ome nucleotides can alsobe used within modified RNA-silencing agents moieties of the instantinvention. Additional modified residues include, deoxy-abasic, inosine,N3-methyl-uridine, N6,N6-dimethyl-adenosine, pseudouridine, purineribonucleoside and ribavirin. In a particularly exemplary embodiment,the 2′ moiety is a methyl group such that the linking moiety is a2′-O-methyl oligonucleotide.

In an exemplary embodiment, the RNA silencing agent of the inventioncomprises locked nucleic acids (LNAs). LNAs comprise sugar-modifiednucleotides that resist nuclease activities (are highly stable) andpossess single nucleotide discrimination for mRNA (Elmen et al., NucleicAcids Res., (2005), 33(1): 439-447; Braasch et al. (2003) Biochemistry42:7967-7975, Petersen et al. (2003) Trends Biotechnol 21:74-81). Thesemolecules have 2′-O,4′-C-ethylene-bridged nucleic acids, with possiblemodifications such as 2′-deoxy-2″-fluorouridine. Moreover, LNAs increasethe specificity of oligonucleotides by constraining the sugar moietyinto the 3′-endo conformation, thereby pre-organizing the nucleotide forbase pairing and increasing the melting temperature of theoligonucleotide by as much as 10° C. per base.

In another exemplary embodiment, the RNA silencing agent of theinvention comprises peptide nucleic acids (PNAs). PNAs comprise modifiednucleotides in which the sugar-phosphate portion of the nucleotide isreplaced with a neutral 2-amino ethylglycine moiety capable of forming apolyamide backbone which is highly resistant to nuclease digestion andimparts improved binding specificity to the molecule (Nielsen, et al.,Science, (2001), 254: 1497-1500).

In certain exemplary embodiments nucleobase-modified ribonucleotides,i.e., ribonucleotides, containing at least one non-naturally occurringnucleobase instead of a naturally occurring nucleobase, are used. Basesmay be modified to block the activity of adenosine deaminase. Exemplarymodified nucleobases include, but are not limited to, uridine and/orcytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine,5-bromo uridine; adenosine and/or guanosines modified at the 8 position,e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O-and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. Itshould be noted that the above modifications may be combined.

In other embodiments, cross-linking can be employed to alter thepharmacokinetics of the RNA silencing agent, for example, to increasehalf-life in the body. Thus, the invention includes RNA silencing agentshaving two complementary strands of nucleic acid, wherein the twostrands are crosslinked. The invention also includes RNA silencingagents which are conjugated or unconjugated (e.g., at its 3′ terminus)to another moiety (e.g. a non-nucleic acid moiety such as a peptide), anorganic compound (e.g., a dye), or the like). Modifying siRNAderivatives in this way may improve cellular uptake or enhance cellulartargeting activities of the resulting siRNA derivative as compared tothe corresponding siRNA, are useful for tracing the siRNA derivative inthe cell, or improve the stability of the siRNA derivative compared tothe corresponding siRNA.

Other exemplary modifications include: (a) 2′ modification, e.g.,provision of a 2′ OMe moiety on a U in a sense or antisense strand, butespecially on a sense strand, or provision of a 2′ OMe moiety in a 3′overhang, e.g., at the 3′ terminus (3′ terminus means at the 3′ atom ofthe molecule or at the most 3′ moiety, e.g., the most 3′ P or 2′position, as indicated by the context); (b) modification of thebackbone, e.g., with the replacement of an 0 with an S, in the phosphatebackbone, e.g., the provision of a phosphorothioate modification, on theU or the A or both, especially on an antisense strand; e.g., with thereplacement of a P with an S; (c) replacement of the U with a C5 aminolinker; (d) replacement of an A with a G (in most cases, sequencechanges are located on the sense strand and not the antisense strand);and (d) modification at the 2′, 6′, 7′, or 8′ position. Exemplaryembodiments are those in which one or more of these modifications arepresent on the sense but not the antisense strand, or embodiments wherethe antisense strand has fewer of such modifications. Yet otherexemplary modifications include the use of a methylated P in a 3′overhang, e.g., at the 3′ terminus; combination of a 2′ modification,e.g., provision of a 2′ O Me moiety and modification of the backbone,e.g., with the replacement of a P with an S, e.g., the provision of aphosphorothioate modification, or the use of a methylated P, in a 3′overhang, e.g., at the 3′ terminus; modification with a 3′ alkyl;modification with an abasic pyrrolidone in a 3′ overhang, e.g., at the3′ terminus; modification with naproxen, ibuprofen, or other moietieswhich inhibit degradation at the 3′ terminus.

4) Modifications to Enhance Cellular Uptake

In other embodiments, RNA silencing agents may be modified with chemicalmoieties, for example, to enhance cellular uptake by target cells (e.g.,neuronal cells). Thus, the invention includes RNA silencing agents whichare conjugated or unconjugated (e.g., at its 3′ terminus) to anothermoiety (e.g. a non-nucleic acid moiety such as a peptide), an organiccompound (e.g., a dye), or the like. The conjugation can be accomplishedby methods known in the art, e.g., using the methods of Lambert et al.,Drug Deliv. Rev.: 47(1), 99-112 (2001) (describes nucleic acids loadedto polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al., J.Control Release 53(1-3):137-43 (1998) (describes nucleic acids bound tonanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4:55-8 (1994)(describes nucleic acids linked to intercalating agents, hydrophobicgroups, polycations or PACA nanoparticles); and Godard et al., Eur. J.Biochem. 232(2):404-10 (1995) (describes nucleic acids linked tonanoparticles).

In a particular embodiment, a modification to the RNA silencing agentsof the invention comprise a vinyl phosphonate (VP) motif in one or moreintersubunit linkers of an oligonucleotide, wherein the VP motif has thefollowing formula:

In a particular embodiment, an RNA silencing agent of invention isconjugated to a lipophilic moiety. In one embodiment, the lipophilicmoiety is a ligand that includes a cationic group. In anotherembodiment, the lipophilic moiety is attached to one or both strands ofan siRNA. In an exemplary embodiment, the lipophilic moiety is attachedto one end of the sense strand of the siRNA. In another exemplaryembodiment, the lipophilic moiety is attached to the 3′ end of the sensestrand. In certain embodiments, the lipophilic moiety is selected fromthe group consisting of cholesterol, vitamin E, vitamin K, vitamin A,folic acid, or a cationic dye (e.g., Cy3). In an exemplary embodiment,the lipophilic moiety is a cholesterol. Other lipophilic moietiesinclude cholic acid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

5) Tethered Ligands

Other entities can be tethered to an RNA silencing agent of theinvention. For example, a ligand tethered to an RNA silencing agent toimprove stability, hybridization thermodynamics with a target nucleicacid, targeting to a particular tissue or cell-type, or cellpermeability, e.g., by an endocytosis-dependent or -independentmechanism. Ligands and associated modifications can also increasesequence specificity and consequently decrease off-site targeting. Atethered ligand can include one or more modified bases or sugars thatcan function as intercalators. In certain exemplary embodiments, theseare located in an internal region, such as in a bulge of RNA silencingagent/target duplex. The intercalator can be an aromatic, e.g., apolycyclic aromatic or heterocyclic aromatic compound. A polycyclicintercalator can have stacking capabilities, and can include systemswith 2, 3, or 4 fused rings. The universal bases described herein can beincluded on a ligand. In one embodiment, the ligand can include acleaving group that contributes to target gene inhibition by cleavage ofthe target nucleic acid. The cleaving group can be, for example, ableomycin (e.g., bleomycin-A5, bleomycin-A2, or bleomycin-B2), pyrene,phenanthroline (e.g., O-phenanthroline), a polyamine, a tripeptide(e.g., lys-tyr-lys tripeptide), or metal ion chelating group. The metalion chelating group can include, e.g., an Lu(III) or EU(III) macrocycliccomplex, a Zn(II) 2,9-dimethylphenanthroline derivative, a Cu(II)terpyridine, or acridine, which can promote the selective cleavage oftarget RNA at the site of the bulge by free metal ions, such as Lu(III).In some embodiments, a peptide ligand can be tethered to a RNA silencingagent to promote cleavage of the target RNA, e.g., at the bulge region.For example, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (cyclam)can be conjugated to a peptide (e.g., by an amino acid derivative) topromote target RNA cleavage. A tethered ligand can be an aminoglycosideligand, which can cause an RNA silencing agent to have improvedhybridization properties or improved sequence specificity. Exemplaryaminoglycosides include glycosylated polylysine, galactosylatedpolylysine, neomycin B, tobramycin, kanamycin A, and acridine conjugatesof aminoglycosides, such as Neo-N-acridine, Neo-S-acridine,Neo-C-acridine, Tobra-N-acridine, and KanaA-N-acridine. Use of anacridine analog can increase sequence specificity. For example, neomycinB has a high affinity for RNA as compared to DNA, but lowsequence-specificity. An acridine analog, neo-5-acridine has anincreased affinity for the HIV Rev-response element (RRE). In someembodiments the guanidine analog (the guanidinoglycoside) of anaminoglycoside ligand is tethered to an RNA silencing agent. In aguanidinoglycoside, the amine group on the amino acid is exchanged for aguanidine group. Attachment of a guanidine analog can enhance cellpermeability of an RNA silencing agent. A tethered ligand can be apoly-arginine peptide, peptoid or peptidomimetic, which can enhance thecellular uptake of an oligonucleotide agent.

Exemplary ligands are coupled, typically covalently, either directly orindirectly via an intervening tether, to a ligand-conjugated carrier. Inexemplary embodiments, the ligand is attached to the carrier via anintervening tether. In exemplary embodiments, a ligand alters thedistribution, targeting or lifetime of an RNA silencing agent into whichit is incorporated. In exemplary embodiments, a ligand provides anenhanced affinity for a selected target, e.g., molecule, cell or celltype, compartment, e.g., a cellular or organ compartment, tissue, organor region of the body, as, e.g., compared to a species absent such aligand.

Exemplary ligands can improve transport, hybridization, and specificityproperties and may also improve nuclease resistance of the resultantnatural or modified RNA silencing agent, or a polymeric moleculecomprising any combination of monomers described herein and/or naturalor modified ribonucleotides. Ligands in general can include therapeuticmodifiers, e.g., for enhancing uptake; diagnostic compounds or reportergroups e.g., for monitoring distribution; cross-linking agents;nuclease-resistance conferring moieties; and natural or unusualnucleobases. General examples include lipophiles, lipids, steroids(e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g.,sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid),vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal),carbohydrates, proteins, protein binding agents, integrin targetingmolecules, polycationics, peptides, polyamines, and peptide mimics.Ligands can include a naturally occurring substance, (e.g., human serumalbumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate(e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin orhyaluronic acid); amino acid, or a lipid. The ligand may also be arecombinant or synthetic molecule, such as a synthetic polymer, e.g., asynthetic polyamino acid. Examples of polyamino acids include polyaminoacid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied)copolymer, divinyl ether-maleic anhydride copolymer,N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol(PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllicacid), N-isopropylacrylamide polymers, or polyphosphazine. Example ofpolyamines include: polyethylenimine, polylysine (PLL), spermine,spermidine, polyamine, pseudopeptide-polyamine, peptidomimeticpolyamine, dendrimer polyamine, arginine, amidine, protamine, cationiclipid, cationic porphyrin, quaternary salt of a polyamine, or an alphahelical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine, multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide orRGD peptide mimetic. Other examples of ligands include dyes,intercalating agents (e.g. acridines and substituted acridines),cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4,texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine, phenanthroline, pyrenes), lys-tyr-lystripeptide, aminoglycosides, guanidium aminoglycodies, artificialendonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol (andthio analogs thereof), cholic acid, cholanic acid, lithocholic acid,adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,glycerol (e.g., esters (e.g., mono, bis, or tris fatty acid esters,e.g., C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, or C₂₀ fattyacids) and ethers thereof, e.g., C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇,C₁₈, C₁₉, or C₂₀ alkyl; e.g., 1,3-bis-O(hexadecyl)glycerol,1,3-bis-O(octaadecyl)glycerol), geranyloxyhexyl group,hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group,palmitic acid, stearic acid (e.g., glyceryl distearate), oleic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, naproxen, vitamin E,folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu³⁺complexes of tetraazamacrocycles), dinitrophenyl, HRP or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, or multivalent fucose. Theligand can be, for example, a lipopolysaccharide, an activator of p38MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the RNA silencing agent into the cell, for example, bydisrupting the cell's cytoskeleton, e.g., by disrupting the cell'smicrotubules, microfilaments, and/or intermediate filaments. The drugcan be, for example, taxon, vincristine, vinblastine, cytochalasin,nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A,indanocine, or myoservin. The ligand can increase the uptake of the RNAsilencing agent into the cell by activating an inflammatory response,for example. Exemplary ligands that would have such an effect includetumor necrosis factor alpha (TNFα), interleukin-1 beta, or gammainterferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule typically binds a serum protein, e.g.,human serum albumin (HSA). An HSA binding ligand allows for distributionof the conjugate to a target tissue, e.g., a non-kidney target tissue ofthe body. For example, the target tissue can be the liver, includingparenchymal cells of the liver. Other molecules that can bind HSA canalso be used as ligands. For example, naproxen or aspirin can be used. Alipid or lipid-based ligand can (a) increase resistance to degradationof the conjugate, (b) increase targeting or transport into a target cellor cell membrane, and/or (c) can be used to adjust binding to a serumprotein, e.g., HSA. A lipid based ligand can be used to modulate, e.g.,control the binding of the conjugate to a target tissue. In a particularembodiment, the lipid based ligand binds HSA. However, it is desiredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed. In another exemplary embodiment, the lipid based ligand bindsHSA weakly or not at all.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HSA and low density lipoprotein (LDL).

In another aspect, the ligand is a cell-permeation agent, typically ahelical cell-permeation agent. In certain exemplary embodiments, theagent is amphipathic. An exemplary agent is a peptide such as tat orantennopedia. If the agent is a peptide, it can be modified, including apeptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages,and use of D-amino acids. The helical agent is typically analpha-helical agent, which typically has a lipophilic face and alipophobic face.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics tooligonucleotide agents can affect pharmacokinetic distribution of theRNA silencing agent, such as by enhancing cellular recognition andabsorption. The peptide or peptidomimetic moiety can be about 5-50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long. A peptide or peptidomimetic can be, for example, a cellpermeation peptide, cationic peptide, amphipathic peptide, orhydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). Thepeptide moiety can be a dendrimer peptide, constrained peptide orcrosslinked peptide. The peptide moiety can be an L-peptide orD-peptide. In another alternative, the peptide moiety can include ahydrophobic membrane translocation sequence (MTS). A peptide orpeptidomimetic can be encoded by a random sequence of DNA, such as apeptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature354:82-84, 1991). In exemplary embodiments, the peptide orpeptidomimetic tethered to an RNA silencing agent via an incorporatedmonomer unit is a cell targeting peptide such as anarginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptidemoiety can range in length from about 5 amino acids to about 40 aminoacids. The peptide moieties can have a structural modification, such asto increase stability or direct conformational properties. Any of thestructural modifications described below can be utilized.

6) Hydrophobic Moieties

In certain embodiments of the double-stranded RNAs provided herein, theRNA molecule is conjugated to one or more hydrophobic moieties (see PCTPub. No. WO 2018/031933, which is incorporated herein by reference). Inan embodiment, the hydrophobic moiety has an affinity for low densitylipoprotein and/or intermediate density lipoprotein. In a relatedembodiment, the hydrophobic moiety is a saturated or unsaturated moietyhaving fewer than three double bonds.

In another embodiment, the hydrophobic moiety has an affinity for highdensity lipoprotein. In a related embodiment, the hydrophobic moiety isa polyunsaturated moiety having at three or more double bonds (e.g.,having three, four, five, six, seven, eight, nine or ten double bonds).In a particular embodiment, the hydrophobic moiety is a polyunsaturatedmoiety having three double bonds. In a particular embodiment, thehydrophobic moiety is a polyunsaturated moiety having four double bonds.In a particular embodiment, the hydrophobic moiety is a polyunsaturatedmoiety having five double bonds. In a particular embodiment, thehydrophobic moiety is a polyunsaturated moiety having six double bonds.

In another embodiment, the hydrophobic moiety is selected from the groupconsisting of fatty acids, steroids, secosteroids, lipids, gangliosidesand nucleoside analogs, and endocannabinoids.

In another embodiment, the hydrophobic moiety is a neuromodulatorylipid, e.g., an endocannabinoid. Non-limiting examples ofendocannabinoids include: anandamide, arachidonoylethanolamine,2-Arachidonyl glyceryl ether (noladin ether), 2-Arachidonyl glycerylether (noladin ether), 2-Arachidonoylglycerol, and N-Arachidonoyldopamine.

In another embodiment, the hydrophobic moiety is an omega-3 fatty acid.Non-limiting examples of omega-3 fatty acids include, but are notlimited to: hexadecatrienoic acid (HTA), alpha-linolenic acid (ALA),stearidonic acid (SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid(ETA), eicosapentaenoic acid (EPA, Timnodonic acid), heneicosapentaenoicacid (HPA), docosapentaenoic acid (DPA, clupanodonic acid),docosahexaenoic acid (DHA, cervonic acid), tetracosapentaenoic acid, andtetracosahexaenoic acid (nisinic acid).

In another embodiment, the hydrophobic moiety is an omega-6 fatty acid.Non-limiting examples of omega-6 fatty acids include, but are notlimited to: linoleic acid, gamma-linolenic acid (GLA), eicosadienoicacid, dihomo-gamma-linolenic acid (DGLA), arachidonic acid (AA),docosadienoic acid, adrenic acid, docosapentaenoic acid (osbond acid),tetracosatetraenoic acid, and tetracosapentaenoic acid.

In another embodiment, the hydrophobic moiety is an omega-9 fatty acid.Non-limiting examples of omega-9 fatty acids include, but are notlimited to: oleic acid, eicosenoic acid, Mead acid, erucic acid, andnervonic acid.

In another embodiment, the hydrophobic moiety is a conjugated linolenicacid. Non-limiting examples of conjugated linolenic acids include, butare not limited to: α-calendic acid, β-calendic acid, jacaric acid,α-eleostearic acid, β-eleostearic acid, catalpic acid, and punicic acid.

In another embodiment, the hydrophobic moiety is a saturated fatty acid.Non-limiting examples of saturated fatty acids include, but are notlimited to: caprylic acid, capric acid, docosanoic acid, lauric acid,myristic acid, palmitic acid, stearic acid, arachidic acid, behenicacid, lignoceric acid, and cerotic acid.

In another embodiment, the hydrophobic moiety is an acid selected fromthe group consisting of: rumelenic acid, α-parinaric acid, β-parinaricacid, bosseopentaenoic acid, pinolenic acid, and podocarpic acid.

In another embodiment, the hydrophobic moiety is selected from the groupconsisting of: docosanoic acid (DCA), docosahexaenoic acid (DHA), andeicosapentaenoic acid (EPA). In a particular embodiment, the hydrophobicmoiety is docosanoic acid (DCA). In another particular embodiment, thehydrophobic moiety is DHA. In another particular embodiment, thehydrophobic moiety is EPA.

In another embodiment, the hydrophobic moiety is a secosteroid. In aparticular embodiment, the hydrophobic moiety is calciferol. In anotherembodiment, the hydrophobic moiety is a steroid other than cholesterol.

In a particular embodiment, the hydrophobic moiety is not cholesterol.

In another embodiment, the hydrophobic moiety is an alkyl chain, avitamin, a peptide, or a bioactive conjugate, including but not limitedto: glycosphingolipids, polyunsaturated fatty acids, secosteroids,steroid hormones, or sterol lipids.

In an embodiment, a double-stranded RNA provided herein comprises one ormore chemically-modified nucleotides. In a particular embodiment, thedouble-stranded RNA comprises alternating 2′-methoxy-nucleotides and2′-fluoro-nucleotides. In another particular embodiment, one or morenucleotides of the double-stranded RNA are connected to adjacentnucleotides via phosphorothioate linkages. In certain embodiments of thedsRNAs disclosed herein, the mismatch nucleotide and the nucleotide(s)adjacent to the mismatch nucleotide are 2′-methoxy-ribonucleotides.

In another particular embodiment, the nucleotides at positions 1 and 2from the 3′ end of the double-stranded RNAs provided herein areconnected to adjacent nucleotides via phosphorothioate linkages. In yetanother particular embodiment, the nucleotides at positions 1 and 2 fromthe 3′ end of the double-stranded RNAs and the nucleotides at positions1 and 2 from the 5′ end of the double-stranded RNAs are connected toadjacent nucleotides via phosphorothioate linkages.

In one embodiment of a double-stranded RNA, the first oligonucleotidecomprises at least 16 contiguous nucleotides, a 5′ end, a 3′ end, andhas complementarity to a target, wherein:

(1) the first oligonucleotide comprises alternating2′-methoxy-nucleotides and 2′-fluoro-nucleotides;

(2) the nucleotides at positions 2 and 14 from the 5′ end are not2′-methoxy-nucleotides;

(3) the nucleotides are connected via phosphodiester or phosphorothioatelinkages; and

(4) the nucleotides at positions 1-6 from the 3′ end, or positions 1-7from the 3′ end, are connected to adjacent nucleotides viaphosphorothioate linkages.

7) Advanced Stabilization Pattern

In one embodiment of the double-stranded RNAs provided herein:

(1) the first oligonucleotide comprises alternating2′-methoxy-ribonucleotides and 2′-fluoro-ribonucleotides, wherein eachnucleotide is a 2′-methoxy-ribonucleotide or a 2′-fluoro-ribonucleotide;and the nucleotides at positions 2 and 14 from the 5′ end of the firstoligonucleotide are not 2′-methoxy-ribonucleotides;

(2) the second oligonucleotide comprises alternating2′-methoxy-ribonucleotides and 2′-fluoro-ribonucleotides, wherein eachnucleotide is a 2′-methoxy-ribonucleotide or a 2′-fluoro-ribonucleotide;and the nucleotides at positions 2 and 14 from the 5′ end of the secondoligonucleotide are 2′-methoxy-ribonucleotides;

(3) the nucleotides of the first oligonucleotide are connected toadjacent nucleotides via phosphodiester or phosphorothioate linkages,wherein the nucleotides at positions 1-6 from the 3′ end, or positions1-7 from the 3′ end are connected to adjacent nucleotides viaphosphorothioate linkages; and

(4) the nucleotides of the second oligonucleotide are connected toadjacent nucleotides via phosphodiester or phosphorothioate linkages,wherein the nucleotides at positions 1 and 2 from the 3′ end areconnected to adjacent nucleotides via phosphorothioate linkages.

In one embodiment of the double-stranded RNAs, the first oligonucleotidehas 3-7 more ribonucleotides than the second oligonucleotide.

In one embodiment, the double-stranded RNA comprises 11-16 base pairduplexes, wherein the nucleotides of each base pair duplex havedifferent chemical modifications (e.g., one nucleotide has a 2′-fluoromodification and the other nucleotide has a 2′-methoxy).

In one embodiment of the double-stranded RNAs, the first oligonucleotidehas 3-7 more ribonucleotides than the second oligonucleotide. In anotherembodiment.

In one embodiment, the first oligonucleotide is the antisense strand andthe second oligonucleotide is the sense strand. See PCT Pub. No. WO2016/161388, which is incorporated herein by reference.

In one embodiment, the first or second oligonucleotide comprises one ormore VP intersubunit modifications having the following formula:

8) Branched Oligonucleotides

Two or more RNA silencing agents as disclosed above, for exampleoligonucleotide constructs such as siRNAs, may be connected to oneanother by one or more moieties independently selected from a linker, aspacer and a branching point, forming a branched oligonucleotidecontaining two or more RNA silencing agents. FIG. 31 illustrates anexemplary di-siRNA di-branched scaffolding for delivering two siRNAs. Inrepresentative embodiments, the nucleic acids of the branchedoligonucleotide each comprise an antisense strand (or portions thereof),wherein the antisense strand has sufficient complementary to aheterozygous single nucleotide polymorphism to mediate an RNA-mediatedsilencing mechanism (e.g. RNAi). In other embodiments, there is provideda second type of branched oligonucleotides featuring nucleic acids thatcomprise a sense strand (or portions thereof) for silencing antisensetranscripts, where the sense strand has sufficient complementarity to anantisense transcript to mediate an RNA-mediated silencing mechanism. Infurther embodiments, there is provided a third type of branchedoligonucleotides including nucleic acids of both types, that is, anucleic acid comprising an antisense strand (or portions thereof) and anoligonucleotide comprising a sense strand (or portions thereof).

In exemplary embodiments, the branched oligonucleotides may have two toeight RNA silencing agents attached through a linker. The linker may behydrophobic. In a particular embodiment, branched oligonucleotides ofthe present application have two to three oligonucleotides. In oneembodiment, the oligonucleotides independently have substantial chemicalstabilization (e.g., at least 40% of the constituent bases arechemically-modified). In a particular embodiment, the oligonucleotideshave full chemical stabilization (i.e., all of the constituent bases arechemically-modified). In some embodiments, branched oligonucleotidescomprise one or more single-stranded phosphorothioated tails, eachindependently having two to twenty nucleotides. In a particularembodiment, each single-stranded tail has eight to ten nucleotides.

In certain embodiments, branched oligonucleotides are characterized bythree properties: (1) a branched structure, (2) full metabolicstabilization, and (3) the presence of a single-stranded tail comprisingphosphorothioate linkers. In a specific embodiment, branchedoligonucleotides have 2 or 3 branches. It is believed that the increasedoverall size of the branched structures promotes increased uptake. Also,without being bound by a particular theory of activity, multipleadjacent branches (e.g., 2 or 3) are believed to allow each branch toact cooperatively and thus dramatically enhance rates ofinternalization, trafficking and release.

Branched oligonucleotides are provided in various structurally diverseembodiments. As shown in FIG. 36 , for example, in some embodimentsnucleic acids attached at the branching points are single stranded andconsist of miRNA inhibitors, gapmers, mixmers, SSOs, PMOs, or PNAs.These single strands can be attached at their 3′ or 5′ end. Combinationsof siRNA and single stranded oligonucleotides could also be used fordual function. In another embodiment, short nucleic acids complementaryto the gapmers, mixmers, miRNA inhibitors, SSOs, PMOs, and PNAs are usedto carry these active single-stranded nucleic acids and enhancedistribution and cellular internalization. The short duplex region has alow melting temperature (T_(m)˜37° C.) for fast dissociation uponinternalization of the branched structure into the cell.

As shown in FIG. 37 , Di-siRNA branched oligonucleotides may comprisechemically diverse conjugates. Conjugated bioactive ligands may be usedto enhance cellular specificity and to promote membrane association,internalization, and serum protein binding. Examples of bioactivemoieties to be used for conjugation include DHAg2, DHA, GalNAc, andcholesterol. These moieties can be attached to Di-siRNA either throughthe connecting linker or spacer, or added via an additional linker orspacer attached to another free siRNA end.

The presence of a branched structure improves the level of tissueretention in the brain more than 100-fold compared to non-branchedcompounds of identical chemical composition, suggesting a new mechanismof cellular retention and distribution. Branched oligonucleotides haveunexpectedly uniform distribution throughout the spinal cord and brain.Moreover, branched oligonucleotides exhibit unexpectedly efficientsystemic delivery to a variety of tissues, and very high levels oftissue accumulation.

Branched oligonucleotides comprise a variety of therapeutic nucleicacids, including ASOs, miRNAs, miRNA inhibitors, splice switching, PMOs,PNAs. In some embodiments, branched oligonucleotides further compriseconjugated hydrophobic moieties and exhibit unprecedented silencing andefficacy in vitro and in vivo.

Linkers

In an embodiment of the branched oligonucleotide, each linker isindependently selected from an ethylene glycol chain, an alkyl chain, apeptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, anester, an amide, a triazole, and combinations thereof; wherein anycarbon or oxygen atom of the linker is optionally replaced with anitrogen atom, bears a hydroxyl substituent, or bears an oxosubstituent. In one embodiment, each linker is an ethylene glycol chain.In another embodiment, each linker is an alkyl chain. In anotherembodiment, each linker is a peptide. In another embodiment, each linkeris RNA. In another embodiment, each linker is DNA. In anotherembodiment, each linker is a phosphate. In another embodiment, eachlinker is a phosphonate. In another embodiment, each linker is aphosphoramidate. In another embodiment, each linker is an ester. Inanother embodiment, each linker is an amide. In another embodiment, eachlinker is a triazole. In another embodiment, each linker is a structureselected from the formulas of FIG. 37 .

9) Compound of Formula (I)

In another aspect, provided herein is a branched oligonucleotidecompound of formula (I):L-(N)_(n)  (I)

wherein L is selected from an ethylene glycol chain, an alkyl chain, apeptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, anester, an amide, a triazole, and combinations thereof, wherein formula(I) optionally further comprises one or more branch point B, and one ormore spacer S; wherein B is independently for each occurrence apolyvalent organic species or derivative thereof; S is independently foreach occurrence selected from an ethylene glycol chain, an alkyl chain,a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, anester, an amide, a triazole, and combinations thereof; N is an RNAduplex comprising a sense strand and an antisense strand, wherein theantisense strand comprises a region of complementarity which issubstantially complementary to a region of a gene comprising an allelicpolymorphism, wherein the antisense strand comprises: a singlenucleotide polymorphism (SNP) position nucleotide at a position 2 to 7from the 5′ end that is complementary to the allelic polymorphism; and amismatch (MM) position nucleotide located 2-11 nucleotides from the SNPposition nucleotide that is a mismatch with a nucleotide in the gene. Inexemplary embodiments, the SNP position nucleotide is at a position 2, 4or 6 from the 5′ end and the mismatch (MM) position nucleotide islocated 2-6 nucleotides from the SNP position nucleotide.

The sense strand and antisense strand each independently comprise one ormore chemical modifications; and n is 2, 3, 4, 5, 6, 7 or 8.

In an embodiment, the compound of formula (I) has a structure selectedfrom formulas (I-1)-(I-9) of Table 1.

TABLE 1 N—L—N (I-1) N—S—L—S—N (I-2)

(I-3)

(I-4)

(I-5)

(I-6)

(I-7)

(I-8)

(I-9)

In one embodiment, the compound of formula (I) is formula (I-1). Inanother embodiment, the compound of formula (I) is formula (I-2). Inanother embodiment, the compound of formula (I) is formula (I-3). Inanother embodiment, the compound of formula (I) is formula (I-4). Inanother embodiment, the compound of formula (I) is formula (I-5). Inanother embodiment, the compound of formula (I) is formula (I-6). Inanother embodiment, the compound of formula (I) is formula (I-7). Inanother embodiment, the compound of formula (I) is formula (I-8). Inanother embodiment, the compound of formula (I) is formula (I-9).

In an embodiment of the compound of formula (I), each linker isindependently selected from an ethylene glycol chain, an alkyl chain, apeptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, anester, an amide, a triazole, and combinations thereof; wherein anycarbon or oxygen atom of the linker is optionally replaced with anitrogen atom, bears a hydroxyl substituent, or bears an oxosubstituent. In one embodiment of the compound of formula (I), eachlinker is an ethylene glycol chain. In another embodiment, each linkeris an alkyl chain. In another embodiment of the compound of formula (I),each linker is a peptide. In another embodiment of the compound offormula (I), each linker is RNA. In another embodiment of the compoundof formula (I), each linker is DNA. In another embodiment of thecompound of formula (I), each linker is a phosphate. In anotherembodiment, each linker is a phosphonate. In another embodiment of thecompound of formula (I), each linker is a phosphoramidate. In anotherembodiment of the compound of formula (I), each linker is an ester. Inanother embodiment of the compound of formula (I), each linker is anamide. In another embodiment of the compound of formula (I), each linkeris a triazole. In another embodiment of the compound of formula (I),each linker is a structure selected from the formulas of FIG. 36 .

In one embodiment of the compound of formula (I), B is a polyvalentorganic species. In another embodiment of the compound of formula (I), Bis a derivative of a polyvalent organic species. In one embodiment ofthe compound of formula (I), B is a triol or tetrol derivative. Inanother embodiment, B is a tri- or tetra-carboxylic acid derivative. Inanother embodiment, B is an amine derivative. In another embodiment, Bis a tri- or tetra-amine derivative. In another embodiment, B is anamino acid derivative. In another embodiment of the compound of formula(I), B is selected from the formulas of FIG. 38 .

Polyvalent organic species are moieties comprising carbon and three ormore valencies (i.e., points of attachment with moieties such as S, L orN, as defined above). Non-limiting examples of polyvalent organicspecies include triols (e.g., glycerol, phloroglucinol, and the like),tetrols (e.g., ribose, pentaerythritol, 1,2,3,5-tetrahydroxybenzene, andthe like), tri-carboxylic acids (e.g., citric acid,1,3,5-cyclohexanetricarboxylic acid, trimesic acid, and the like),tetra-carboxylic acids (e.g., ethylenediaminetetraacetic acid,pyromellitic acid, and the like), tertiary amines (e.g.,tripropargylamine, triethanolamine, and the like), triamines (e.g.,diethylenetriamine and the like), tetramines, and species comprising acombination of hydroxyl, thiol, amino, and/or carboxyl moieties (e.g.,amino acids such as lysine, serine, cysteine, and the like).

In an embodiment of the compound of formula (I), each nucleic acidcomprises one or more chemically-modified nucleotides. In an embodimentof the compound of formula (I), each nucleic acid consists ofchemically-modified nucleotides. In certain embodiments of the compoundof formula (I), >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%or >50% of each nucleic acid comprises chemically-modified nucleotides.

In an embodiment, each antisense strand independently comprises a 5′terminal group R selected from the groups of Table 2:

TABLE 2

R¹

R²

R³

R⁴

R⁵

R⁶

R⁷

R⁸

In one embodiment, R is R₁. In another embodiment, R is R₂. In anotherembodiment, R is R₃. In another embodiment, R is R₄. In anotherembodiment, R is R₅. In another embodiment, R is R₆. In anotherembodiment, R is R₇. In another embodiment, R is R₈.

Structure of Formula (II)

In an embodiment, the compound of formula (I) the structure of formula(II):

wherein X, for each occurrence, independently, is selected fromadenosine, guanosine, uridine, cytidine, and chemically-modifiedderivatives thereof; Y, for each occurrence, independently, is selectedfrom adenosine, guanosine, uridine, cytidine, and chemically-modifiedderivatives thereof; - represents a phosphodiester internucleosidelinkage; = represents a phosphorothioate internucleoside linkage; and--- represents, individually for each occurrence, a base-pairinginteraction or a mismatch.

In certain embodiments, the structure of formula (II) does not containmismatches. In one embodiment, the structure of formula (II) contains 1mismatch. In another embodiment, the compound of formula (II) contains 2mismatches. In another embodiment, the compound of formula (II) contains3 mismatches. In another embodiment, the compound of formula (II)contains 4 mismatches. In an embodiment, each nucleic acid consists ofchemically-modified nucleotides.

In certainembodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%or >50% of X's of the structure of formula (II) are chemically-modifiednucleotides. In otherembodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%or >50% of X's of the structure of formula (II) are chemically-modifiednucleotides.

Structure of Formula (III)

In an embodiment, the compound of formula (I) has the structure offormula (III):

wherein X, for each occurrence, independently, is a nucleotidecomprising a 2′-deoxy-2′-fluoro modification; X, for each occurrence,independently, is a nucleotide comprising a 2′-O-methyl modification; Y,for each occurrence, independently, is a nucleotide comprising a2′-deoxy-2′-fluoro modification; and Y, for each occurrence,independently, is a nucleotide comprising a 2′-O-methyl modification.

In an embodiment, X is chosen from the group consisting of2′-deoxy-2′-fluoro modified adenosine, guanosine, uridine or cytidine.In an embodiment, X is chosen from the group consisting of 2′-O-methylmodified adenosine, guanosine, uridine or cytidine. In an embodiment, Yis chosen from the group consisting of 2′-deoxy-2′-fluoro modifiedadenosine, guanosine, uridine or cytidine. In an embodiment, Y is chosenfrom the group consisting of 2′-O-methyl modified adenosine, guanosine,uridine or cytidine.

In certain embodiments, the structure of formula (III) does not containmismatches. In one embodiment, the structure of formula (III) contains 1mismatch. In another embodiment, the compound of formula (III) contains2 mismatches. In another embodiment, the compound of formula (III)contains 3 mismatches. In another embodiment, the compound of formula(III) contains 4 mismatches.

Structure of Formula (IV)

In an embodiment, the compound of formula (I) has the structure offormula (IV):

wherein X, for each occurrence, independently, is selected fromadenosine, guanosine, uridine, cytidine, and chemically-modifiedderivatives thereof; Y, for each occurrence, independently, is selectedfrom adenosine, guanosine, uridine, cytidine, and chemically-modifiedderivatives thereof; - represents a phosphodiester internucleosidelinkage; = represents a phosphorothioate internucleoside linkage; and--- represents, individually for each occurrence, a base-pairinginteraction or a mismatch.

In certain embodiments, the structure of formula (IV) does not containmismatches. In one embodiment, the structure of formula (IV) contains 1mismatch. In another embodiment, the compound of formula (IV) contains 2mismatches. In another embodiment, the compound of formula (IV) contains3 mismatches. In another embodiment, the compound of formula (IV)contains 4 mismatches. In an embodiment, each nucleic acid consists ofchemically-modified nucleotides.

In certainembodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%or >50% of X's of the structure of formula (II) are chemically-modifiednucleotides. In otherembodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%or >50% of X's of the structure of formula (II) are chemically-modifiednucleotides.

Structure of Formula (V)

In an embodiment, the compound of formula (I) has the structure offormula (V):

wherein X, for each occurrence, independently, is a nucleotidecomprising a 2′-deoxy-2′-fluoro modification; X, for each occurrence,independently, is a nucleotide comprising a 2′-O-methyl modification; Y,for each occurrence, independently, is a nucleotide comprising a2′-deoxy-2′-fluoro modification; and Y, for each occurrence,independently, is a nucleotide comprising a 2′-O-methyl modification.

In certain embodiments, X is chosen from the group consisting of2′-deoxy-2′-fluoro modified adenosine, guanosine, uridine or cytidine.In an embodiment, X is chosen from the group consisting of 2′-O-methylmodified adenosine, guanosine, uridine or cytidine. In an embodiment, Yis chosen from the group consisting of 2′-deoxy-2′-fluoro modifiedadenosine, guanosine, uridine or cytidine. In an embodiment, Y is chosenfrom the group consisting of 2′-O-methyl modified adenosine, guanosine,uridine or cytidine.

In certain embodiments, the structure of formula (V) does not containmismatches. In one embodiment, the structure of formula (V) contains 1mismatch. In another embodiment, the compound of formula (V) contains 2mismatches. In another embodiment, the compound of formula (V) contains3 mismatches. In another embodiment, the compound of formula (V)contains 4 mismatches.

Variable Linkers

In an embodiment of the compound of formula (I), L has the structure ofL1:

In an embodiment of L1, R is R³ and n is 2.

In an embodiment of the structure of formula (II), L has the structureof L1. In an embodiment of the structure of formula (III), L has thestructure of L1. In an embodiment of the structure of formula (IV), Lhas the structure of L1. In an embodiment of the structure of formula(V), L has the structure of L1. In an embodiment of the structure offormula (VI), L has the structure of L1. In an embodiment of thestructure of formula (VI), L has the structure of L1.

In an embodiment of the compound of formula (I), L has the structure ofL2:

In an embodiment of L2, R is R³ and n is 2. In an embodiment of thestructure of formula (II), L has the structure of L2. In an embodimentof the structure of formula (III), L has the structure of L2. In anembodiment of the structure of formula (IV), L has the structure of L2.In an embodiment of the structure of formula (V), L has the structure ofL2. In an embodiment of the structure of formula (VI), L has thestructure of L2. In an embodiment of the structure of formula (VI), Lhas the structure of L2.

10) Delivery System

In a further aspect, provided herein is a delivery system fortherapeutic nucleic acids having the structure of formula (VI):L-(cNA)_(n)  (VI)wherein L is selected from an ethylene glycol chain, an alkyl chain, apeptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, anester, an amide, a triazole, and combinations thereof, wherein formula(VI) optionally further comprises one or more branch point B, and one ormore spacer S; wherein B is independently for each occurrence apolyvalent organic species or derivative thereof; S is independently foreach occurrence selected from an ethylene glycol chain, an alkyl chain,a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, anester, an amide, a triazole, and combinations thereof; each cNA,independently, is a carrier nucleic acid comprising one or more chemicalmodifications; and n is 2, 3, 4, 5, 6, 7 or 8.

In one embodiment of the delivery system, L is an ethylene glycol chain.In another embodiment of the delivery system, L is an alkyl chain. Inanother embodiment of the delivery system, L is a peptide. In anotherembodiment of the delivery system, L is RNA. In another embodiment ofthe delivery system, L is DNA. In another embodiment of the deliverysystem, L is a phosphate. In another embodiment of the delivery system,L is a phosphonate. In another embodiment of the delivery system, L is aphosphoramidate. In another embodiment of the delivery system, L is anester. In another embodiment of the delivery system, L is an amide. Inanother embodiment of the delivery system, L is a triazole.

In one embodiment of the delivery system, S is an ethylene glycol chain.In another embodiment, S is an alkyl chain. In another embodiment of thedelivery system, S is a peptide. In another embodiment, S is RNA. Inanother embodiment of the delivery system, S is DNA. In anotherembodiment of the delivery system, S is a phosphate. In anotherembodiment of the delivery system, S is a phosphonate. In anotherembodiment of the delivery system, S is a phosphoramidate. In anotherembodiment of the delivery system, S is an ester. In another embodiment,S is an amide. In another embodiment, S is a triazole.

In one embodiment of the delivery system, n is 2. In another embodimentof the delivery system, n is 3. In another embodiment of the deliverysystem, n is 4. In another embodiment of the delivery system, n is 5. Inanother embodiment of the delivery system, n is 6. In another embodimentof the delivery system, n is 7. In another embodiment of the deliverysystem, n is 8.

In certain embodiments, each cNAcomprises >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55% or >50%chemically-modified nucleotides.

In an embodiment, the compound of formula (VI) has a structure selectedfrom formulas (VI-1)-(VI-9) of Table 3:

TABLE 3 ANc—L—cNA (VI-1) ANc—S—L—S—cNA (VI-2)

(VI-3)

(VI-4)

(VI-5)

(VI-6)

(VI-7)

(VI-8)

(VI-9)

In an embodiment, the compound of formula (VI) is the structure offormula (VI-1). In an embodiment, the compound of formula (VI) is thestructure of formula (VI-2). In an embodiment, the compound of formula(VI) is the structure of formula (VI-3). In an embodiment, the compoundof formula (VI) is the structure of formula (VI-4). In an embodiment,the compound of formula (VI) is the structure of formula (VI-5). In anembodiment, the compound of formula (VI) is the structure of formula(VI-6). In an embodiment, the compound of formula (VI) is the structureof formula (VI-7). In an embodiment, the compound of formula (VI) is thestructure of formula (VI-8). In an embodiment, the compound of formula(VI) is the structure of formula (VI-9).

In an embodiment, the compound of formulas (VI) (including, e.g.,formulas (VI-1)-(VI-9), each cNA independently comprises at least 15contiguous nucleotides. In an embodiment, each cNA independentlyconsists of chemically-modified nucleotides.

In an embodiment, the delivery system further comprises n therapeuticnucleic acids (NA), wherein each NA comprises a region ofcomplementarity which is substantially complementary to a region of agene comprising an allelic polymorphism, wherein the antisense strandcomprises: a single nucleotide polymorphism (SNP) position nucleotide ata position 2 to 7 from the 5′ end that is complementary to the allelicpolymorphism; and a mismatch (MM) position nucleotide located 2-11nucleotide from the SNP position nucleotide that is a mismatch with anucleotide in the gene. In exemplary embodiments, the SNP positionnucleotide is at a position 2, 4 or 6 from the 5′ end and the mismatch(MM) position nucleotide is located 2-6 nucleotides from the SNPposition nucleotide. Also, each NA is hybridized to at least one cNA. Inone embodiment, the delivery system is comprised of 2 NAs. In anotherembodiment, the delivery system is comprised of 3 NAs. In anotherembodiment, the delivery system is comprised of 4 NAs. In anotherembodiment, the delivery system is comprised of 5 NAs. In anotherembodiment, the delivery system is comprised of 6 NAs. In anotherembodiment, the delivery system is comprised of 7 NAs. In anotherembodiment, the delivery system is comprised of 8 NAs.

In an embodiment, each NA independently comprises at least 16 contiguousnucleotides. In an embodiment, each NA independently comprises 16-20contiguous nucleotides. In an embodiment, each NA independentlycomprises 16 contiguous nucleotides. In another embodiment, each NAindependently comprises 17 contiguous nucleotides. In anotherembodiment, each NA independently comprises 18 contiguous nucleotides.In another embodiment, each NA independently comprises 19 contiguousnucleotides. In another embodiment, each NA independently comprises 20contiguous nucleotides.

In an embodiment, each NA comprises an unpaired overhang of at least 2nucleotides. In another embodiment, each NA comprises an unpairedoverhang of at least 3 nucleotides. In another embodiment, each NAcomprises an unpaired overhang of at least 4 nucleotides. In anotherembodiment, each NA comprises an unpaired overhang of at least 5nucleotides. In another embodiment, each NA comprises an unpairedoverhang of at least 6 nucleotides. In an embodiment, the nucleotides ofthe overhang are connected via phosphorothioate linkages.

In an embodiment, each NA, independently, is selected from the groupconsisting of: DNA, siRNAs, antagomiRs, miRNAs, gapmers, mixmers, orguide RNAs. In one embodiment, each NA, independently, is a DNA. Inanother embodiment, each NA, independently, is a siRNA. In anotherembodiment, each NA, independently, is an antagomiR. In anotherembodiment, each NA, independently, is a miRNA. In another embodiment,each NA, independently, is a gapmer. In another embodiment, each NA,independently, is a mixmer. In another embodiment, each NA,independently, is a guide RNA. In an embodiment, each NA is the same. Inan embodiment, each NA is not the same.

In an embodiment, the delivery system further comprising n therapeuticnucleic acids (NA) has a structure selected from formulas (I), (II),(III), (IV), (V), (VI), and embodiments thereof described herein. In oneembodiment, the delivery system has a structure selected from formulas(I), (II), (III), (IV), (V), (VI), and embodiments thereof describedherein further comprising 2 therapeutic nucleic acids (NA). In anotherembodiment, the delivery system has a structure selected from formulas(I), (II), (III), (IV), (V), (VI), and embodiments thereof describedherein further comprising 3 therapeutic nucleic acids (NA). In oneembodiment, the delivery system has a structure selected from formulas(I), (II), (III), (IV), (V), (VI), and embodiments thereof describedherein further comprising 4 therapeutic nucleic acids (NA). In oneembodiment, the delivery system has a structure selected from formulas(I), (II), (III), (IV), (V), (VI), and embodiments thereof describedherein further comprising 5 therapeutic nucleic acids (NA). In oneembodiment, the delivery system has a structure selected from formulas(I), (II), (III), (IV), (V), (VI), and embodiments thereof describedherein further comprising 6 therapeutic nucleic acids (NA). In oneembodiment, the delivery system has a structure selected from formulas(I), (II), (III), (IV), (V), (VI), and embodiments thereof describedherein further comprising 7 therapeutic nucleic acids (NA). In oneembodiment, the delivery system has a structure selected from formulas(I), (II), (III), (IV), (V), (VI), and embodiments thereof describedherein further comprising 8 therapeutic nucleic acids (NA).

In one embodiment, the delivery system has a structure selected fromformulas (I), (II), (III), (IV), (V), (VI), further comprising a linkerof structure L1 or L2 wherein R is R3 and n is 2. In another embodiment,the delivery system has a structure selected from formulas (I), (II),(III), (IV), (V), (VI), further comprising a linker of structure L1wherein R is R3 and n is 2. In another embodiment, the delivery systemhas a structure selected from formulas (I), (II), (III), (IV), (V),(VI), further comprising a linker of structure L2 wherein R is R3 and nis 2.

Pharmaceutical Compositions and Methods of Administration

In one aspect, provided herein is a pharmaceutical compositioncomprising a therapeutically effective amount of one or more compound,oligonucleotide, or nucleic acid as described herein, and apharmaceutically acceptable carrier. In one embodiment, thepharmaceutical composition comprises one or more double-stranded,chemically-modified nucleic acid as described herein, and apharmaceutically acceptable carrier. In a particular embodiment, thepharmaceutical composition comprises one double-stranded,chemically-modified nucleic acid as described herein, and apharmaceutically acceptable carrier. In another particular embodiment,the pharmaceutical composition comprises two double-stranded,chemically-modified nucleic acids as described herein, and apharmaceutically acceptable carrier.

In a particular embodiment, the pharmaceutical composition comprises adouble-stranded RNA molecule comprising about 15-35 nucleotidescomplementary to a region of a gene encoding a heterozygous SNP mutantprotein, said region comprising an allelic polymorphism, and a secondstrand comprising about 15-35 nucleotides complementary to the firststrand, wherein the dsRNA molecule comprises a mismatch that is not inthe position of the allelic polymorphism; and the mismatch and thenucleotide corresponding to the polymorphism are not in the center ofthe dsRNA molecule.

In an embodiment, the mismatch is 4 nucleotides upstream, 3 nucleotidesupstream nucleotide corresponding to the allelic polymorphism, 2nucleotides upstream nucleotide corresponding to the allelicpolymorphism, 1 nucleotide upstream, 1 nucleotide downstream nucleotidecorresponding to the allelic polymorphism, 2 nucleotides downstreamnucleotide corresponding to the allelic polymorphism, 3 nucleotidesdownstream nucleotide corresponding to the allelic polymorphism, 4nucleotides downstream nucleotide corresponding to the allelicpolymorphism, or 5 nucleotides downstream nucleotide corresponding tothe allelic polymorphism. In certain embodiments, the mismatch is notadjacent to the nucleotide corresponding to the allelic polymorphism.

In another embodiment of the pharmaceutical composition, thedouble-stranded RNA comprises a nucleotide corresponding to the allelicpolymorphism which is in position 2, 3, 4, 5, or 6 from the 5′ end. Inan embodiment, the nucleotide corresponding to the allelic polymorphismis in position 2 from the 5′ end. In an embodiment, the nucleotidecorresponding to the allelic polymorphism is in position 3 from the 5′end. In an embodiment, the nucleotide corresponding to the allelicpolymorphism is in position 4 from the 5′ end. In an embodiment, thenucleotide corresponding to the allelic polymorphism is in position 5from the 5′ end. In an embodiment, the nucleotide corresponding to theallelic polymorphism is in position 6 from the 5′ end.

In an embodiment of the pharmaceutical composition, the double-strandedRNA selectively silences a mutant allele having an allelic polymorphism,e.g., a heterozygous SNP. In an embodiment of the pharmaceuticalcomposition, the double-stranded RNA silences a mutant allele having anallelic polymorphism and does not affect the wild-type allele of thesame gene. In another embodiment of the pharmaceutical composition, thedouble-stranded RNA provided herein silences a mutant allele having anallelic polymorphism and silences the wild-type allele of the same geneto a lesser extent than the mutant allele.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous (IV),intradermal, subcutaneous (SC or SQ), intraperitoneal, intramuscular,oral (e.g., inhalation), transdermal (topical), and transmucosaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfate; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be desired to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, typical methods of preparation are vacuumdrying and freeze-drying which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds should typically lie within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the EC50 (i.e., the concentration ofthe test compound which achieves a half-maximal response) as determinedin cell culture. Such information can be used to more accuratelydetermine useful doses in humans. Levels in plasma may be measured, forexample, by high performance liquid chromatography.

Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a diseaseor disorder caused, in whole or in part, by an allelic polymorphism(e.g., a heterozygous SNP). In one embodiment, the disease or disorderis a trinucleotide repeat disease or disorder. In another embodiment,the disease or disorder is a polyglutamine disorder. In an embodiment,the methods comprise administering a therapeutically effective amount ofa double-stranded RNA molecule provided herein. In an embodiment, thedisease or disorder is a disorder associated with the expression ofhuntingtin and in which alteration of huntingtin, especially theamplification of CAG repeat copy number, leads to a defect in thehuntingtin gene (structure or function) or huntingtin protein (structureor function or expression), such that clinical manifestations includethose seen in Huntington's disease patients.

In embodiments of the methods, the double-stranded RNAs disclosed hereinare homologous to an allelic polymorphism except for one mismatchedoligonucleotide at a particular position relative to the nucleotidecorresponding to the allelic polymorphism. In certain embodiments, themismatch is within about 6 nucleotides of the nucleotide correspondingto the allelic polymorphism, within about 5 nucleotides of thenucleotide corresponding to the allelic polymorphism, within about 4nucleotides of the nucleotide corresponding to the allelic polymorphismwithin about 3 nucleotide of the nucleotide corresponding to the allelicpolymorphism, within about 2 nucleotide of the nucleotide correspondingto the allelic polymorphism, or within about 1 nucleotides of thenucleotide corresponding to the allelic polymorphism. In particularlyexemplary embodiments, the mismatch is not adjacent to the nucleotidecorresponding to the allelic polymorphism.

In another embodiment of the methods, the double-stranded RNA comprisesa nucleotide corresponding to the allelic polymorphism which is inposition 2, 3, 4, 5, or 6 from the 5′ end. In an embodiment, thenucleotide corresponding to the allelic polymorphism is in position 2from the 5′ end. In an embodiment, the nucleotide corresponding to theallelic polymorphism is in position 3 from the 5′ end. In an embodiment,the nucleotide corresponding to the allelic polymorphism is in position4 from the 5′ end. In an embodiment, the nucleotide corresponding to theallelic polymorphism is in position 5 from the 5′ end. In an embodiment,the nucleotide corresponding to the allelic polymorphism is in position6 from the 5′ end.

In an embodiment of the methods, the dsRNA comprises a nucleotidecorresponding to a polymorphism at position 6 from the 5′ end and amismatch at position 11 from the 5′ end. In an embodiment of themethods, the dsRNA comprises a nucleotide corresponding to apolymorphism at position 4 from the 5′ end and a mismatch at position 7from the 5′ end.

In another embodiment of the methods, the double-stranded RNAselectively silences a mutant allele having an allelic polymorphism. Inan embodiment, the double-stranded RNA silences a mutant allele havingan allelic polymorphism and does not affect the wild-type allele of thesame gene. In another embodiment, the double-stranded RNA silences amutant allele having an allelic polymorphism and silences the wild-typeallele of the same gene to a lesser extent than the mutant allele.

In an embodiment of the methods, the dsRNA comprises one or more VPintersubunit linkage modifications wherein the intersubunit linkage hasthe following formula:

In additional embodiments, the dsRNA comprises one or more of theintersubunit linkage modifications depicted in FIG. 43 .

“Treatment,” or “treating,” as used herein, is defined as theapplication or administration of a therapeutic agent (e.g., a RNA agentor vector or transgene encoding same) to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has the disease or disorder, a symptom of disease ordisorder or a predisposition toward a disease or disorder, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease or disorder, the symptoms of the diseaseor disorder, or the predisposition toward disease.

In one aspect, the invention provides a method for preventing in asubject, a disease or disorder as described above, by administering tothe subject a therapeutic agent (e.g., an RNAi agent or vector ortransgene encoding same). Subjects at risk for the disease can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe disease or disorder, such that the disease or disorder is preventedor, alternatively, delayed in its progression.

Another aspect of the invention pertains to methods treating subjectstherapeutically, i.e., alter onset of symptoms of the disease ordisorder. In an exemplary embodiment, the modulatory method of theinvention involves contacting a cell expressing a gain-of-functionmutant with a therapeutic agent (e.g., a RNAi agent or vector ortransgene encoding same) that is specific for one or more targetsequences within the gene, such that sequence specific interference withthe gene is achieved. These methods can be performed in vitro (e.g., byculturing the cell with the agent) or, alternatively, in vivo (e.g., byadministering the agent to a subject).

An RNA silencing agent modified for enhanced uptake into neural cellscan be administered at a unit dose less than about 1.4 mg per kg ofbodyweight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005,0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight, andless than 200 nmole of RNA agent (e.g., about 4.4×10¹⁶ copies) per kg ofbodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75,0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075 or 0.00015 nmole of RNAsilencing agent per kg of bodyweight. The unit dose, for example, can beadministered by injection (e.g., intravenous or intramuscular,intrathecally, or directly into the brain), an inhaled dose, or atopical application. In exemplary embodiments, dosages are less than 2,1 or 0.1 mg/kg of body weight.

Delivery of an RNA silencing agent directly to an organ (e.g., directlyto the brain) can be at a dosage on the order of about 0.00001 mg toabout 3 mg per organ, or about 0.0001-0.001 mg per organ, about 0.03-3.0mg per organ, about 0.1-3.0 mg per organ or about 0.3-3.0 mg per organ.The dosage can be an amount effective to treat or prevent a neurologicaldisease or disorder (e.g., Huntington's disease). In one embodiment, theunit dose is administered less frequently than once a day, e.g., lessthan every 2, 4, 8 or 30 days. In another embodiment, the unit dose isnot administered with a frequency (e.g., not a regular frequency). Forexample, the unit dose may be administered a single time. In oneembodiment, the effective dose is administered with other traditionaltherapeutic modalities.

In one embodiment, a subject is administered an initial dose, and one ormore maintenance doses of an RNA silencing agent. The maintenance doseor doses are generally lower than the initial dose, e.g., one-half lessof the initial dose. A maintenance regimen can include treating thesubject with a dose or doses ranging from 0.01 μg to 1.4 mg/kg of bodyweight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg ofbodyweight per day. The maintenance doses are typically administered nomore than once every 5, 10, or 30 days. Further, the treatment regimenmay last for a period of time which will vary depending upon the natureof the particular disease, its severity and the overall condition of thepatient. In particular embodiments, the dosage may be delivered no morethan once per day, e.g., no more than once per 24, 36, 48 or more hours,e.g., no more than once every 5 or 8 days. Following treatment, thepatient can be monitored for changes in his condition and foralleviation of the symptoms of the disease state. The dosage of thecompound may either be increased in the event the patient does notrespond significantly to current dosage levels, or the dose may bedecreased if an alleviation of the symptoms of the disease state isobserved, if the disease state has been ablated, or if undesiredside-effects are observed.

Huntington's Disease

In certain aspects of the invention, RNA silencing agents are designedto target polymorphisms (e.g., heterozygous single nucleotidepolymorphisms) in the mutant human huntingtin protein (htt) for thetreatment of Huntington's disease. Accordingly, in another aspect,provided herein is a method of treating or managing Huntington's diseasecomprising administering to a patient in need of such treatment ormanagement a therapeutically effective amount of a compound,oligonucleotide, or nucleic acid as described herein, or apharmaceutical composition comprising said compound, oligonucleotide, ornucleic acid.

Huntington's disease, inherited as an autosomal dominant disease, causesimpaired cognition and motor disease. Patients can live more than adecade with severe debilitation, before premature death from starvationor infection. The disease begins in the fourth or fifth decade for mostcases, but a subset of patients manifest disease in teenage years. Thegenetic mutation for Huntington's disease is a lengthened CAG repeat inthe huntingtin gene. The CAG repeat varies in number from 8 to 35 copiesin normal individuals (Kremer et al., 1994). The genetic mutation (e.g.,an increase in length of the CAG repeats from less than 36 in the normalhuntingtin gene to greater than 36 in the disease) is associated withthe synthesis of a mutant huntingtin protein, which has greater than 36consecutive polyglutamine residues (Aronin et al., 1995). In general,individuals with 36 or more CAG repeats will get Huntington's disease.Prototypic for as many as twenty other diseases with a lengthened CAG asthe underlying mutation, Huntington's disease still has no effectivetherapy. A variety of interventions—such as interruption of apoptoticpathways, addition of reagents to boost mitochondrial efficiency, andblockade of NMDA receptors—have shown promise in cell cultures and mousemodel of Huntington's disease. However, at best these approaches reveala short prolongation of cell or animal survival.

The disease gene linked to Huntington's disease is termed Huntingtin or(htt). The huntingtin locus is large, spanning 180 kb and consisting of67 exons. The huntingtin gene is widely expressed and is required fornormal development. It is expressed as 2 alternatively polyadenylatedforms displaying different relative abundance in various fetal and adulttissues. The larger transcript is approximately 13.7 kb and is expressedpredominantly in adult and fetal brain whereas the smaller transcript ofapproximately 10.3 kb is more widely expressed. The two transcriptsdiffer with respect to their 3′ untranslated regions (Lin et al., 1993).Both messages are predicted to encode a 348 kilodalton proteincontaining 3144 amino acids. The genetic defect leading to Huntington'sdisease is believed to confer a new property on the mRNA or alter thefunction of the protein.

Huntington's disease complies with the central dogma of genetics: amutant gene serves as a template for production of a mutant mRNA; themutant mRNA then directs synthesis of a mutant protein (Aronin et al.,1995; DiFiglia et al., 1997). Mutant huntingtin (protein) likelyaccumulates in selective neurons in the striatum and cortex, disrupts asyet determined cellular activities, and causes neuronal dysfunction anddeath (Aronin et al., 1999; Laforet et al., 2001). Because a single copyof a mutant gene suffices to cause Huntington's disease, the mostparsimonious treatment would render the mutant gene ineffective.Theoretical approaches might include stopping gene transcription ofmutant huntingtin, destroying mutant mRNA, and blocking translation.Each has the same outcome—loss of mutant huntingtin.

Huntington SNPs

Exemplary SNPs in the huntingtin gene sequence suitable for targetingaccording to certain exemplary embodiments are disclosed in Table 4below. Genomic sequence for each SNP site can be found in, for example,the publicly available “SNP Entrez” database maintained by the NCBI. Thefrequency of heterozygosity for each SNP site for HD patient and controlDNA is further illustrated in Table 4. Targeting combinations offrequently heterozygous SNPs allows the treatment of a large percentageof the individuals in a HD population using a relatively small number ofallele-specific RNA silencing agents.

TABLE 4 htt SNPs. rs363125 ORF, 11.00% GTTAAGAGATGGGGACAGT exon 39A[A/C]TTCAACGCTAGAA GAACACA (SEQ ID NO: 1) rs362273 ORF, 35.20%AGCCACGAGAAGCTGCTGC exon 57 T[A/G]CAGATCAACCCCG AGCGGGA (SEQ ID NO: 2)rs362307 3′ UTR, 48.60% CCGGAGCCTTTGGAAGTCT exon 67 G[C/T]GCCCTTGTGCCCTGCCTCCA (SEQ ID NO: 3) rs362336 ORF, 37.40% CAGCCCGAGCTGCCTGCAG exon 48A[A/G]CCGGCGGCCTACT GGAGCAA (SEQ ID NO: 4) rs362331 ORF, 39.40%CCCACGCCTGCTCCCTCAT exon 50 C[C/T]ACTGTGTGCACTT CATCCTG (SEQ ID NO: 5)rs362272 ORF, 36.10% GGGTTGGAGCCCTGCACGG exon 61 C[A/G]TCCTCTATGTGCTGGAGTGC (SEQ ID NO: 6) rs362306 3′ UTR, 35.80% CTGCTGGTTGTTGCCAGGTexon 67 T[A/G]CAGCTGCTCTTGC ATCTGGG (SEQ ID NO: 7) rs362268 3′ UTR,35.80% TCCTCCCTCCTGCAGGCTG exon 67 G[C/G]TGTTGGCCCCTSTGCTGTCC (SEQ ID NO: 8) rs362267 3′ UTR, 35.50% GATTTGGGAGCTCTGCTTGexon 67 C[C/T]GACTGGCTGTGAG ACGAGGC (SEQ ID NO: 9) rs363099 ORF, 35.80%GAAAAGTTTGGAGGGTTTC exon 29 T[C/T]CGCTCAGCCTTGG ATGTTCT (SEQ ID NO: 10)

In one embodiment, RNA silencing agents of the invention are capable oftargeting one or more of the SNP sites listed in Table 4. In oneembodiment, RNA silencing agents of the invention are capable oftargeting rs363125 SNP site of the Huntingtin mRNA. In anotherembodiment, RNA silencing agents of the invention are capable oftargeting rs362273 SNP site of the Huntingtin mRNA. In anotherembodiment, RNA silencing agents of the invention are capable oftargeting rs362307 SNP site of the Huntingtin mRNA. In anotherembodiment, RNA silencing agents of the invention are capable oftargeting rs362336 SNP site of the Huntingtin mRNA. In anotherembodiment, RNA silencing agents of the invention are capable oftargeting rs362331 SNP site of the Huntingtin mRNA. In anotherembodiment, RNA silencing agents of the invention are capable oftargeting rs362272 SNP site of the Huntingtin mRNA. In anotherembodiment, RNA silencing agents of the invention are capable oftargeting rs362306 SNP site of the Huntingtin mRNA. In anotherembodiment, RNA silencing agents of the invention are capable oftargeting rs362268 SNP site of the Huntingtin mRNA. In anotherembodiment, RNA silencing agents of the invention are capable oftargeting rs362267 SNP site of the Huntingtin mRNA. In anotherembodiment, RNA silencing agents of the invention are capable oftargeting rs363099 SNP site of the Huntingtin mRNA. In some embodiments,SNP sites targeted by RNA silencing agents are associated withHuntington's Disease. In particularly exemplary embodiments, SNP sitestargeted by RNA silencing agents are significantly associated withHuntington's Disease.

In additional exemplary embodiments, the RNA silencing agents includeone or more of the sequences of Tables 5-7:

TABLE 5 additional compound snp mismatch sequence HTT SNP name positionposition antisense strand SEQ ID NO: sense strand SEQ ID NO: rs362273SNP2-7 2 7 UUAGCAUCAGCUUCUCGUGG 230 AGAAGCUGCUGCUAA 242 rs362273 SNP4-74 7 UUGUAGUAGCAGCUUCUCGU 231 AAGCUGCUGCUACAA 243 rs362273 SNP4-8 4 8UUGUAGCUGCAGCUUCUCGU 232 AAGCUGCUGCUACAA 243 rs362273 SNP4-15 4 15UUGUAGCAGCAGCUACUCGU 233 AAGCUGCUGCUACAA 243 rs362273 SNP6-5A 6 5UUCUAUAGCAGCAGCUUCUC 234 GCUGCUGCUACAGAA 244 rs362273 SNP6-8 6 8UUCUGUAUCAGCAGCUUCUC 235 GCUGCUGCUACAGAA 244 rs362273 SNP6-11 6 11UUCUGUAGCAUCAGCUUCUC 236 GCUGCUGCUACAGAA 244 rs362273 SNP6-14 6 14UUCUGUAGCAGCAUCUUCUC 237 GCUGCUGCUACAGAA 244 rs362273 SNP6-16 6 16UUCUGUAGCAGCAGCAUCUC 238 GCUGCUGCUACAGAA 244 rs362307 SNP3-5G 3 5UCGCGGACUUCCAAAGGCUC 239 UUUGGAAGUCCGCGA 245 rs362307 SNP3-7G 3 7UCGCAGGCUUCCAAAGGCUC 240 UUUGGAAGCCUGCGA 246 rs362307 SNP3-8 3 8UCGCAGAUUUCCAAAGGCUC 241 UUUGGAAAUCUGCGA 247 In DNA molecules, U can bereplaced with T

TABLE 6 description of siRNA sequence flanking snp site additionalsequence SNP of interest compound snp  mismatch SEQ ID SEQ ID  SNP nameposition position antisense strand  NO: sense strand NO: rs362273 (A)SNP2-7 2 7 UUAGCAUCAGCUUCUCGUGG 230 AGAAGCUGCUGCUAA 242 rs362273 (A)SNP4-7 4 7 UUGUAGUAGCAGCUUCUCGU 231 AAGCUGCUGCUACAA 243 rs362273 (A)SNP4-8 4 8 UUGUAGCUGCAGCUUCUCGU 232 AAGCUGCUGCUACAA 243 rs362273 (A)SNP4-15 4 15 UUGUAGCAGCAGCUACUCGU 233 AAGCUGCUGCUACAA 243 rs362273 (A)SNP6-5A 6 5 UUCUAUAGCAGCAGCUUCUC 234 GCUGCUGCUACAGAA 244 rs362273 (A)SNP6-8 6 8 UUCUGUAUCAGCAGCUUCUC 235 GCUGCUGCUACAGAA 244 rs362273 (A)SNP6-11 6 11 UUCUGUAGCAUCAGCUUCUC 236 GCUGCUGCUACAGAA 244 rs362273 (A)SNP6-14 6 14 UUCUGUAGCAGCAUCUUCUC 237 GCUGCUGCUACAGAA 244 rs362273 (A)SNP6-16 6 16 UUCUGUAGCAGCAGCAUCUC 238 GCUGCUGCUACAGAA 244 rs362307 (C′)SNP3-5G 3 5 UCGCGGACUUCCAAAGGCUC 239 UUUGGAAGUCCGCGA 245 rs362307 (C′)SNP3-7G 3 7 UCGCAGGCUUCCAAAGGCUC 240 UUUGGAAGCCUGCGA 246 rs362307 (C′)SNP3-8 3 8 UCGCAGAUUUCCAAAGGCUC 241 UUUGGAAAUCUGCGA 247

TABLE 7 description of siRNA sequence flanking snp additional sequenceSNP of interest compound snp mismatch SEQ ID SEQ ID SNP name positionposition antisense strand  NO: sense strand  NO: rs362273 (G) SNP2-7 2 7UCAGCAUCAGCUUCUCGUGG 248 AGAAGCUGCUGCUGA 259 rs362273 (G) SNP4-7 4 7UUGCAGUAGCAGCUUCUCGU 249 AAGCUGCUGCUGCAA 260 rs362273 (G) SNP4-8 4 8UUGCAGCUGCAGCUUCUCGU 250 AAGCUGCUGCUGCAA 260 rs362273 (G) SNP4-15 4 15UUGCAGCAGCAGCUACUCGU 251 AAGCUGCUGCUGCAA 260 rs362273 (G) SNP6-5A 6 5UUCUACAGCAGCAGCUUCUC 252 GCUGCUGCUGCAGAA 261 rs362273 (G) SNP6-8 6 8UUCUGCAUCAGCAGCUUCUC 253 GCUGCUGCUGCAGAA 261 rs362273 (G) SNP6-11 6 11UUCUGCAGCAUCAGCUUCUC 254 GCUGCUGCUGCAGAA 261 rs362273 (G) SNP6-14 6 14UUCUGCAGCAGCAUCUUCUC 255 GCUGCUGCUGCAGAA 261 rs362307 (T) SNP3-5G 3 5UCACGGACUUCCAAAGGCUC 256 UUUGGAAGUCCGUGA 262 rs362307 (T) SNP3-7G 3 7UCACAGGCUUCCAAAGGCUC 257 UUUGGAAGCCUGUGA 263 rs362307 (T) SNP3-8 3 8UCACAGAUUUCCAAAGGCUC 258 UUUGGAAAUCUGUGA 264Methods of Delivering Nucleic Acids

RNA silencing agents of the invention may be directly introduced into acell (e.g., a neural cell) (i.e., intracellularly); or introducedextracellularly into a cavity, interstitial space, into the circulationof an organism, introduced orally, or may be introduced by bathing acell or organism in a solution containing the nucleic acid. Vascular orextravascular circulation, the blood or lymph system, and thecerebrospinal fluid are sites where the nucleic acid may be introduced.

The RNA silencing agents of the invention can be introduced usingnucleic acid delivery methods known in art including injection of asolution containing the nucleic acid, bombardment by particles coveredby the nucleic acid, soaking the cell or organism in a solution of thenucleic acid, or electroporation of cell membranes in the presence ofthe nucleic acid. Other methods known in the art for introducing nucleicacids to cells may be used, such as lipid-mediated carrier transport,chemical-mediated transport, and cationic liposome transfection such ascalcium phosphate, and the like. The nucleic acid may be introducedalong with other components that perform one or more of the followingactivities: enhance nucleic acid uptake by the cell or other-wiseincrease inhibition of the target gene.

Physical methods of introducing nucleic acids include injection of asolution containing the RNA, bombardment by particles covered by theRNA, soaking the cell or organism in a solution of the RNA, orelectroporation of cell membranes in the presence of the RNA. A viralconstruct packaged into a viral particle would accomplish both efficientintroduction of an expression construct into the cell and transcriptionof RNA encoded by the expression construct. Other methods known in theart for introducing nucleic acids to cells may be used, such aslipid-mediated carrier transport, chemical-IS mediated transport, suchas calcium phosphate, and the like. Thus, the RNA may be introducedalong with components that perform one or more of the followingactivities: enhance RNA uptake by the cell, inhibit annealing of singlestrands, stabilize the single strands, or other-wise increase inhibitionof the target gene.

RNA may be directly introduced into the cell (i.e., intracellularly), orintroduced extracellularly into a cavity, interstitial space, into thecirculation of an organism, introduced orally, or may be introduced bybathing a cell or organism in a solution containing the RNA. Vascular orextravascular circulation, the blood or lymph system, and thecerebrospinal fluid are sites where the RNA may be introduced.

The cell having the target gene may be from the germ line or somatic,totipotent or pluripotent, dividing or non-dividing, parenchyma orepithelium, immortalized or transformed, or the like. The cell may be astem cell or a differentiated cell. Cell types that are differentiatedinclude adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium,neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages,neutrophils, eosinophils, basophils, mast cells, leukocytes,granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts,hepatocytes, and cells of the endocrine or exocrine glands.

Depending on the particular target gene and the dose of double-strandedRNA material delivered, this process may provide partial or completeloss of function for the target gene. A reduction or loss of geneexpression in at least 50%, 60%, 70%, 80%, 90%, 95% or 99% or more oftargeted cells is exemplary. Inhibition of gene expression refers to theabsence (or observable decrease) in the level of protein and/or mRNAproduct from a target gene. Specificity refers to the ability to inhibitthe target gene without manifest effects on other genes of the cell. Theconsequences of inhibition can be confirmed by examination of theoutward properties of the cell or organism (as presented below in theexamples) or by biochemical techniques such as RNA solutionhybridization, nuclease protection, Northern hybridization, reversetranscription, gene expression monitoring with a microarray, antibodybinding, enzyme linked immunosorbent assay (ELISA), Western blotting,radioimmunoassay (RIA), other immunoassays, fluorescence activated cellanalysis (FACS) and the like.

For RNA-mediated inhibition in a cell line or whole organism, geneexpression is conveniently assayed by use of a reporter or drugresistance gene whose protein product is easily assayed. Such reportergenes include acetohydroxyacid synthase (AHAS), alkaline phosphatase(AP), beta galactosidase (LacZ), beta glucoronidase (GUS),chloramphenicol acetyltransferase (CAT), green fluorescent protein(GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase(NOS), octopine synthase (OCS), and derivatives thereof. Multipleselectable markers are available that confer resistance to ampicillin,bleomycin, chloramphenicol, gentarnycin, hygromycin, kanamycin,lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin.Depending on the assay, quantitation of the amount of gene expressionallows one to determine a degree of inhibition which is greater than10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treatedaccording to the present invention. Lower doses of injected material andlonger times after administration of RNAi agent may result in inhibitionin a smaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%,or 95% of targeted cells). Quantization of gene expression in a cell mayshow similar amounts of inhibition at the level of accumulation oftarget mRNA or translation of target protein. As an example, theefficiency of inhibition may be determined by assessing the amount ofgene product in the cell. mRNA may be detected with a hybridizationprobe having a nucleotide sequence outside the region used for theinhibitory double-stranded RNA, or translated polypeptide may bedetected with an antibody raised against the polypeptide sequence ofthat region.

The RNA may be introduced in an amount which allows delivery of at leastone copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000copies per cell) of material may yield more effective inhibition; lowerdoses may also be useful for specific applications.

In a particular aspect, the efficacy of an RNAi agent of the invention(e.g., an siRNA targeting a polymorphism in a mutant gene) is tested forits ability to specifically degrade mutant mRNA (e.g., mutant htt mRNAand/or the production of mutant huntingtin protein) in cells, inparticular, in neurons (e.g., striatal or cortical neuronal clonal linesand/or primary neurons). Also suitable for cell-based validation assaysare other readily transfectable cells, for example, HeLa cells or COScells. Cells are transfected with human wild-type or mutant cDNAs (e.g.,human wild-type or mutant huntingtin cDNA). Standard siRNA, modifiedsiRNA or vectors able to produce siRNA from U-looped mRNA areco-transfected. Selective reduction in mutant mRNA (e.g., mutanthuntingtin mRNA) and/or mutant protein (e.g., mutant huntingtin) ismeasured. Reduction of mutant mRNA or protein can be compared to levelsof normal mRNA or protein. Exogenously-introduced normal mRNA or protein(or endogenous normal mRNA or protein) can be assayed for comparisonpurposes. When utilizing neuronal cells, which are known to be somewhatresistant to standard transfection techniques, it may be desirable tointroduce RNAi agents (e.g., siRNAs) by passive uptake.

In certain exemplary embodiments, a composition that includes an RNAagent, e.g., a dsRNA agent, of the invention can be delivered to thenervous system of a subject by a variety of routes. Exemplary routesinclude intrathecal, parenchymal (e.g., in the brain), nasal, and oculardelivery. The composition can also be delivered systemically, e.g., byintravenous, subcutaneous or intramuscular injection, which isparticularly useful for delivery of the RNA agents, e.g., dsRNA agents,to peripheral neurons. An exemplary route of delivery is directly to thebrain, e.g., into the ventricles or the hypothalamus of the brain, orinto the lateral or dorsal areas of the brain. The RNA agents, e.g.,dsRNA agents, for neural cell delivery can be incorporated intopharmaceutical compositions suitable for administration.

For example, compositions can include one or more species of an RNAagent, e.g., a dsRNA agent, and a pharmaceutically acceptable carrier.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, intranasal,transdermal), oral or parenteral. Parenteral administration includesintravenous drip, subcutaneous, intraperitoneal or intramuscularinjection, intrathecal, or intraventricular (e.g.,intracerebroventricular) administration. In certain exemplaryembodiments, an RNA silencing agent of the invention is delivered acrossthe Blood-Brain-Barrier (BBB) suing a variety of suitable compositionsand methods described herein.

The route of delivery can be dependent on the disorder of the patient.For example, a subject diagnosed with Huntington's disease can beadministered an anti-htt RNA agent, e.g., a dsRNA agent, of theinvention directly into the brain (e.g., into the globus pallidus or thecorpus striatum of the basal ganglia, and near the medium spiny neuronsof the corpus striatum). In addition to an RNA silencing agent of theinvention, a patient can be administered a second therapy, e.g., apalliative therapy and/or disease-specific therapy. The secondarytherapy can be, for example, symptomatic (e.g., for alleviatingsymptoms), neuroprotective (e.g., for slowing or halting diseaseprogression), or restorative (e.g., for reversing the disease process).For the treatment of Huntington's disease, for example, symptomatictherapies can include the drugs haloperidol, carbamazepine, orvalproate. Other therapies can include psychotherapy, physiotherapy,speech therapy, communicative and memory aids, social support services,and dietary advice.

An RNA agent, e.g., a dsRNA agent, can be delivered to neural cells ofthe brain. Delivery methods that do not require passage of thecomposition across the blood-brain barrier can be utilized. For example,a pharmaceutical composition containing an RNA agent, e.g., a dsRNAagent, can be delivered to the patient by injection directly into thearea containing the disease-affected cells. For example, thepharmaceutical composition can be delivered by injection directly intothe brain. The injection can be by stereotactic injection into aparticular region of the brain (e.g., the substantia nigra, cortex,hippocampus, striatum, or globus pallidus). The RNA agent, e.g., a dsRNAagent, can be delivered into multiple regions of the central nervoussystem (e.g., into multiple regions of the brain, and/or into the spinalcord). The RNA agent, e.g., a dsRNA agent, can be delivered into diffuseregions of the brain (e.g., diffuse delivery to the cortex of thebrain).

In one embodiment, the RNA agent, e.g., a dsRNA agent, can be deliveredby way of a cannula or other delivery device having one end implanted ina tissue, e.g., the brain, e.g., the substantia nigra, cortex,hippocampus, striatum or globus pallidus of the brain. The cannula canbe connected to a reservoir of RNA agent, e.g., dsRNA agent. The flow ordelivery can be mediated by a pump, e.g., an osmotic pump or minipump,such as an Alzet pump (Durect, Cupertino, Calif.). In one embodiment, apump and reservoir are implanted in an area distant from the tissue,e.g., in the abdomen, and delivery is effected by a conduit leading fromthe pump or reservoir to the site of release. Devices for delivery tothe brain are described, for example, in U.S. Pat. Nos. 6,093,180, and5,814,014.

An RNA agent, e.g., a dsRNA agent, of the invention can be furthermodified such that it is capable of traversing the blood brain barrier.For example, the RNA agent, e.g., a dsRNA agent, can be conjugated to amolecule that enables the agent to traverse the barrier. Such modifiedRNA agents, e.g., dsRNA agents, can be administered by any desiredmethod, such as by intraventricular or intramuscular injection, or bypulmonary delivery, for example.

In certain embodiments, exosomes are used to deliver an RNA agent, e.g.,a dsRNA agent, of the invention. Exosomes can cross the BBB and deliversiRNAs, antisense oligonucleotides, chemotherapeutic agents and proteinsspecifically to neurons after systemic injection (See, Alvarez-Erviti L,Seow Y, Yin H, Betts C, Lakhal S, Wood M J. (2011). Delivery of siRNA tothe mouse brain by systemic injection of targeted exosomes. NatBiotechnol. 2011 April; 29(4):341-5. doi: 10.1038/nbt.1807;El-Andaloussi S, Lee Y, Lakhal-Littleton S, Li J, Seow Y, Gardiner C,Alvarez-Erviti L, Sargent I L, Wood M J. (2011). Exosome-mediateddelivery of siRNA in vitro and in vivo. Nat Protoc. 2012 December;7(12):2112-26. doi: 10.1038/nprot.2012.131; EL Andaloussi S, Mager I,Breakefield X O, Wood M J. (2013). Extracellular vesicles: biology andemerging therapeutic opportunities. Nat Rev Drug Discov. 2013 May;12(5):347-57. doi: 10.1038/nrd3978; El Andaloussi S, Lakhal S, Mäger I,Wood M J. (2013). Exosomes for targeted siRNA delivery across biologicalbarriers. Adv. Drug Deliv Rev. 2013 March; 65(3):391-7. doi:10.1016/j.addr.2012.08.008).

In certain embodiments, one or more lipophilic molecules are used toallow delivery of an RNA agent, e.g., a dsRNA agent, of the inventionpast the BBB (Alvarez-Ervit (2011)). The RNA silencing agent would thenbe activated, e.g., by enzyme degradation of the lipophilic disguise torelease the drug into its active form.

In certain embodiments, one or more receptor-mediated permeabilizingcompounds can be used to increase the permeability of the BBB to allowdelivery of an RNA silencing agent of the invention. These drugsincrease the permeability of the BBB temporarily by increasing theosmotic pressure in the blood which loosens the tight junctions betweenthe endothelial cells ((El-Andaloussi (2012)). By loosening the tightjunctions normal intravenous injection of an RNA silencing agent can beperformed.

In certain embodiments, nanoparticle-based delivery systems are used todeliver an RNA agent, e.g., a dsRNA agent, of the invention across theBBB. As used herein, “nanoparticles” refer to polymeric nanoparticlesthat are typically solid, biodegradable, colloidal systems that havebeen widely investigated as drug or gene carriers (S. P. Egusquiaguirre,M. Igartua, R. M. Hernandez, and J. L. Pedraz, “Nanoparticle deliverysystems for cancer therapy: advances in clinical and preclinicalresearch,” Clinical and Translational Oncology, vol. 14, no. 2, pp.83-93, 2012). Polymeric nanoparticles are classified into two majorcategories, natural polymers and synthetic polymers. Natural polymersfor siRNA delivery include, but are not limited to, cyclodextrin,chitosan, and atelocollagen (Y. Wang, Z. Li, Y. Han, L. H. Liang, and A.Ji, “Nanoparticle-based delivery system for application of siRNA invivo,” Current Drug Metabolism, vol. 11, no. 2, pp. 182-196, 2010).Synthetic polymers include, but are not limited to, polyethyleneimine(PEI), poly(dl-lactide-co-glycolide) (PLGA), and dendrimers, which havebeen intensively investigated (X. Yuan, S. Naguib, and Z. Wu, “Recentadvances of siRNA delivery by nanoparticles,” Expert Opinion on DrugDelivery, vol. 8, no. 4, pp. 521-536, 2011). For a review ofnanoparticles and other suitable delivery systems, See Jong-Min Lee,Tae-Jong Yoon, and Young-Seok Cho, “Recent Developments inNanoparticle-Based siRNA Delivery for Cancer Therapy,” BioMed ResearchInternational, vol. 2013, Article ID 782041, 10 pages, 2013.doi:10.1155/2013/782041 (incorporated by reference in its entirety.)

An RNA agent, e.g., a dsRNA agent, of the invention can be administeredocularly, such as to treat retinal disorder, e.g., a retinopathy. Forexample, the pharmaceutical compositions can be applied to the surfaceof the eye or nearby tissue, e.g., the inside of the eyelid. They can beapplied topically, e.g., by spraying, in drops, as an eyewash, or anointment. Ointments or droppable liquids may be delivered by oculardelivery systems known in the art such as applicators or eye droppers.Such compositions can include mucomimetics such as hyaluronic acid,chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinylalcohol), preservatives such as sorbic acid, EDTA or benzylchroniumchloride, and the usual quantities of diluents and/or carriers. Thepharmaceutical composition can also be administered to the interior ofthe eye, and can be introduced by a needle or other delivery devicewhich can introduce it to a selected area or structure. The compositioncontaining the RNA silencing agent can also be applied via an ocularpatch.

In general, an RNA agent, e.g., a dsRNA agent, of the invention can beadministered by any suitable method. As used herein, topical deliverycan refer to the direct application of an RNA agent, e.g., a dsRNAagent, to any surface of the body, including the eye, a mucous membrane,surfaces of a body cavity, or to any internal surface. Formulations fortopical administration may include transdermal patches, ointments,lotions, creams, gels, drops, sprays, and liquids. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Topical administration can alsobe used as a means to selectively deliver the RNA agent, e.g., a dsRNAagent, to the epidermis or dermis of a subject, or to specific stratathereof, or to an underlying tissue.

Compositions for intrathecal or intraventricular (e.g.,intracerebroventricular) administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Compositions for intrathecal or intraventricularadministration typically do not include a transfection reagent or anadditional lipophilic moiety besides, for example, the lipophilic moietyattached to the RNA agent, e.g., a dsRNA agent.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir. Forintravenous use, the total concentration of solutes should be controlledto render the preparation isotonic.

An RNA agent, e.g., a dsRNA agent, of the invention can be administeredto a subject by pulmonary delivery. Pulmonary delivery compositions canbe delivered by inhalation of a dispersion so that the compositionwithin the dispersion can reach the lung where it can be readilyabsorbed through the alveolar region directly into blood circulation.Pulmonary delivery can be effective both for systemic delivery and forlocalized delivery to treat diseases of the lungs. In one embodiment, anRNA agent, e.g., a dsRNA agent, administered by pulmonary delivery hasbeen modified such that it is capable of traversing the blood brainbarrier.

Pulmonary delivery can be achieved by different approaches, includingthe use of nebulized, aerosolized, micellular and dry powder-basedformulations. Delivery can be achieved with liquid nebulizers,aerosol-based inhalers, and dry powder dispersion devices. Metered-dosedevices are exemplary. One of the benefits of using an atomizer orinhaler is that the potential for contamination is minimized because thedevices are self-contained. Dry powder dispersion devices, for example,deliver drugs that may be readily formulated as dry powders. An RNAsilencing agent composition may be stably stored as lyophilized orspray-dried powders by itself or in combination with suitable powdercarriers. The delivery of a composition for inhalation can be mediatedby a dosing timing element which can include a timer, a dose counter,time measuring device, or a time indicator which when incorporated intothe device enables dose tracking, compliance monitoring, and/or dosetriggering to a patient during administration of the aerosol medicament.

The types of pharmaceutical excipients that are useful as carriersinclude stabilizers such as human serum albumin (HSA), bulking agentssuch as carbohydrates, amino acids and polypeptides; pH adjusters orbuffers; salts such as sodium chloride; and the like. These carriers maybe in a crystalline or amorphous form or may be a mixture of the two.

Bulking agents that are particularly valuable include compatiblecarbohydrates, polypeptides, amino acids or combinations thereof.Suitable carbohydrates include monosaccharides such as galactose,D-mannose, sorbose, and the like; disaccharides, such as lactose,trehalose, and the like; cyclodextrins, such as2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such asraffinose, maltodextrins, dextrans, and the like; alditols, such asmannitol, xylitol, and the like. An exemplary group of carbohydratesincludes lactose, trehalose, raffinose maltodextrins, and mannitol.Suitable polypeptides include aspartame. Amino acids include alanine andglycine, with glycine being exemplary.

Suitable pH adjusters or buffers include organic salts prepared fromorganic acids and bases, such as sodium citrate, sodium ascorbate, andthe like; sodium citrate is exemplary.

An RNA agent, e.g., a dsRNA agent, of the invention can be administeredby oral and nasal delivery. For example, drugs administered throughthese membranes have a rapid onset of action, provide therapeutic plasmalevels, avoid first pass effect of hepatic metabolism, and avoidexposure of the drug to the hostile gastrointestinal (GI) environment.Additional advantages include easy access to the membrane sites so thatthe drug can be applied, localized and removed easily. In oneembodiment, an RNA silencing agent administered by oral or nasaldelivery has been modified to be capable of traversing the blood-brainbarrier.

In one embodiment, unit doses or measured doses of a composition thatinclude RNA agents, e.g., dsRNA agents, are dispensed by an implanteddevice. The device can include a sensor that monitors a parameter withina subject. For example, the device can include a pump, such as anosmotic pump and, optionally, associated electronics.

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods described hereinmay be made using suitable equivalents without departing from the scopeof the embodiments disclosed herein. Having now described certainembodiments in detail, the same will be more clearly understood byreference to the following examples, which are included for purposes ofillustration only and are not intended to be limiting.

EXAMPLES Example 1: SNP Discrimination Varies According to the Positionof the Mismatch

FIG. 46 is a flow chart illustrating a methodology for generating andselecting SNP-discriminating siRNAs that was implemented in the instanceof HTT, but is also applicable to SNPs in other genes. A primary screenis conducted to determine which position the SNP is placed at causes thegreatest discrimination. Then, the mismatch position(s) yielding bestresults are selected, and affinity for non-target alleles is furtherreduced in a secondary screening where chemical and structuraloptimizations to the siRNA molecule with improved selectivity and/orpotency are selected.

There are several SNPs within the HTT gene that have high rates ofheterozygosity in HD patients (FIG. 45 ). For optimization ofSNP-specific RNAi-mediated silencing of huntingtin, SNP rs362273 in exon57 of HTT mRNA was used as model target for optimization of SNPselective silencing. This SNP heterozygosity occurs in 35% of the HDpatient population.

The psiCHECK reporter plasmid described herein contains SNP rs362273 anda partial flanking region from exon 57 of htt, within a Rluc 3′ UTR. Thewild-type psiCHECK reporter plasmid contains the same region of httwithout the SNP (FIG. 1 ).

Hydrophobically modified RNAs (hsiRNAs) designed to be complimentary tothe Huntingtin (htt) mRNA containing the mutant SNP (2273-1 (A)) werescreened for efficacy with the psiCheck reporter plasmid system. Thenumber following SNP represents the position of the SNP in the siRNA(FIG. 47 ). FIG. 2 shows that placing the SNP in position 2, 4 or 6provided the greatest SNP discrimination, without losing efficacyagainst the mutant allele. HeLa cells transfected with one of tworeporter plasmids were reverse transfected with 1.5 μM hsiRNAs bypassive uptake, and treated for 72 hours. Luciferase activity wasmeasured at 72 hours post transfection (FIG. 2 ).

The hsiRNAs were further tested for allelic discrimination in a doseresponse dual luciferase assay in HeLa cells (FIG. 3 ). Multiple hsiRNAspreferentially silenced the reporter plasmid containing the mutant SNPas compared to the wild-type reporter plasmid. HeLa cells transfectedwith one of two reporter plasmids were reverse transfected with 1.5 μMhsiRNAs by passive uptake, and treated for 72 hours. Reporter plasmidexpression was measured at 72 hours post transfection (FIG. 3 ).

Example 2: SNP Discrimination in the Endogenous Htt mRNA

The hsiRNAs were tested for efficacy against the endogenous HuntingtinmRNA containing a homozygous rs362273 SNP. As HeLa cells are homozygousat rs362273, with an A on each allele, allelic discrimination was notassessed with this assay. Instead, FIG. 4 shows that two hsiRNAs, SNP4-0and SNP6-0, were highly effective at silencing the htt mRNA containingthe correct SNP. The mRNA levels were measured using Quantigene 2.0 bDNAassay after treating HeLa cells with hsiRNAs via passive uptake for 72hours. Human htt mRNA levels were normalized to human HPRT.

Example 3: Designing hsiRNAs with a Second Mismatch for Greater AllelicDiscrimination

For each of the three hsiRNAs (SNP2-0, SNP4-0, and SNP6-0, also namedmm2, mm4, and mm6, respectively) previously chosen for dose response, 16new hsiRNAs were designed and synthesized with slight sequencemodifications (FIG. 34 ). These sequences introduced a single mismatchat every possible position along the original sequence, in order to testif the second mismatch impairs silencing of the off-target SNP moresignificantly than before, with little effect on silencing the targetSNP. Antisense strand sequences shown 5′ to 3′, with the SNP site inred, and the new mismatch in blue (FIG. 12 ).

A primary screen of the efficacy of the hsiRNAs in FIG. 12 showed thatthe position of the second mismatch, relative to the position of thenucleotide corresponding to the SNP, resulted in varying levels of SNPdiscrimination in HeLa cells. HeLa cells transfected with one of twopsiCHECK reporter plasmids were reverse transfected with 1.5 μM hsiRNAsby passive uptake, and treated for 72 hours. Luciferase activity wasmeasured at 72 hours post transfection. FIG. 5 shows that multiplehsiRNAs discriminately silenced the reporter plasmid containing the SNPmutation as compared to the wild-type reporter plasmid.

The most efficacious hsiRNAs, containing the second mismatch, werefurther tested in a dose response curve to verify improved SNPdiscrimination. HeLa cells transfected with one of two reporter plasmidswere reverse transfected with hsiRNAs by passive uptake, and treated for72 hours. Reporter expression measured with a dual-luciferase assay.FIGS. 6-8 show the IC50 values of the hsiRNAs with two mismatches forsilencing the reporter plasmid containing the SNP mutation versus thewild-type reporter plasmid. The SNP6-11 hsiRNA (hsiRNA molecule with thenucleotide corresponding to the polymorphism at position 6 from the 5′end and the mismatch at position 11 from the 5′ end) and the SNP4-7hsiRNA (hsiRNA molecule with the nucleotide corresponding to thepolymorphism at position 4 from the 5′ end and the mismatch at position7 from the 5′ end) were shown to be the most efficacious (see FIGS. 7-9). Surprisingly, altering the modification pattern around the SNPrescues efficacy lost by introducing the second mismatch withoutimpairing discrimination. The SNP6-11 hsiRNA was altered so that it had2′O-methyl modifications flanking the mismatch nucleotide (as well asthe mismatch nucleotide itself having the 2′O-methyl modification) (seeFIG. 10 ).

Example 4: Additional Modifications

A variety of oligonucleotide types (e.g., gapmers, mixmers, miRNAinhibitors, splice-switching oligonucleotides (“SSOs”),phosphorodiamidate morpholino oligonucleotides (“PMOs”), peptide nucleicacids (“PNAs”) and the like) can be used in the oligonucleotidesdescribed herein, optionally utilizing various combinations ofmodifications (e.g., chemical modifications) and/or conjugationsdescribed herein and in, e.g., U.S. Ser. No. 15/089,423; U.S. Ser. No.15/236,051; U.S. Ser. No. 15/419,593; U.S. Ser. No. 15/697,120 and U.S.Pat. No. 9,809,817; and U.S. Ser. No. 15/814,350 and U.S. Pat. No.9,862,350, each of which is incorporated herein by reference in itsentirety for all purposes.

For example, an oligonucleotide described herein may be designed as adi-siRNA (see, e.g., FIG. 14 ). An oligonucleotide described herein mayinclude one or more different backbone linkages (see, e.g., FIG. 15 ).An oligonucleotide described herein may include a variety of sugarmodifications (see, e.g., FIG. 16 ). An oligonucleotide described hereinmay include a variety of internucleotide bonds (see, e.g., FIG. 17 ). Anoligonucleotide described herein may include one or more 5′stabilization modifications (see, e.g., FIG. 18 ). An oligonucleotidedescribed herein may include one or more conjugated moieties (see, e.g.,FIG. 19 ). Illustrated in FIG. 35 are a number of exemplaryoligonucleotide backbone modifications.

An oligonucleotide described herein can effectively be used to target aG at the SNP site simply by changing the base at the SNP position. Asseen in FIG. 33 , compound SNP6-11 was synthesized a second time, thistime to target a G at the SNP site instead of an A. This allowed forselectively silencing either allele, a strategy that is very useful forpatients with different heterozygosities at the same SNP site.

In certain exemplary embodiments, one or more abasic nucleotides areutilized at an SNP position nucleotide, at a MM position nucleotide, atthe 5′ end, at the 3′ end, or any combination of these.

In certain exemplary embodiments, hsiRNAs are synthesized with varyingsugar modifications around the mismatch to improve allele specificity,e.g., 2′FANA instead of 2′F; triple 2′F or triple 2′ OMe aroundSNP/mismatch position.

Example 5: HTT Mouse Model

BAC97-HD refer to a transgenic mouse comprising a human bacterialartificial chromosome (BAC) transgenic insert containing the entirepathogenic 170 kb human Huntingtin (htt) genomic locus that was modifiedby replacing the human htt exon 1 with a loxP-flanked human mutant httexon 1 sequence containing 97 mixed CAA-CAG repeats encoding acontinuous polyglutamine (polyQ) stretch.

Lead compound (SNP6-11) was synthesized into the di-branched chemicalscaffold having the structure illustrated in FIG. 31 and subsequentlytested in vivo via 40 nmol bilateral intracerebroventricular (ICV)injection (20 nmols to each side) in BAC97-HD female mice at 8 weeks ofage. The mice had two copies of normal mouse htt gene with a G at SNPrs362273 and a transgenic insert of pathogenic human htt gene with an Aat SNP rs362273A. A nonsense sequence with no target matches in the RNAtranscriptome was also synthesized into the same di-branched scaffoldand injected in the mice as a negative control (NTC).

Several brain regions were collected from the mice for RNA and proteinanalysis 1 month post injection, and HTT protein levels were measured bywestern blot using Ab1 antibody. FIG. 32A is a western blot performed oncollected striatum tissue, and protein levels normalized to vinculin arepresented in FIG. 32B.

Example 6: SNP Targeting is Sequence-Independent

Whether SNP discrimination of lead compounds was sequence-dependent wasassessed. Hydrophobically modified RNAs (hsiRNAs) designed to becomplimentary to the Huntingtin (htt) mRNA containing a U to G mismatchor a C to A mismatch in rs362273 were used. Both the 6-11 hsiRNAcomplementary to a U to G mismatch and the 6-11 hsiRNA complementary toa C to A mismatch preferentially cleaved the target SNP (FIG. 20 ).

Example 7: Synthesis of Vinyl Phosphonate Modified Intersubunit Linkages

Representative syntheses of the vinyl phosphinate modified intersubunitlinkages discussed herein are illustrated in FIGS. 21 and 29 . Thesynthetic procedure of FIG. 21 is detailed below.

Synthesis of Compound 3a

Anhydrous solution of compound 2a (16.6 g, 20.8 mmol) in pyridine (100mL) was added anhydrous DIPEA (6.5 mL, 37.4 mmol) and benzoyl chloride(3.6 mL, 31.2 mmol). After the mixture was stirred for 4 hours at roomtemperature, excess pyridine was evaporated and diluted with CH₂Cl₂. Theorganic solution was washed by sat. aq. NaHCO₃. The organic layer wascollected, dried over MgSO₄, filtered and evaporated. Obtained crudematerial was purified by silica gel column chromatography (hexane-ethylacetate, 4:1 to 1:1) yielding compound 3a as a slightly yellow foam(14.5 g, 78%); ¹H NMR (500 MHz, CDCl₃) δ 7.88-7.87 (m, 2H), 7.84 (d, 1H,J=8.3 Hz), 7.67-7.58 (m, 5H), 7.48-7.45 (m, 4H), 7.39-7.32 (m, 4H),7.25-7.23 (m, 3H), 7.18-7.17 (m, 2H), 7.12-7.07 (m, 4H), 6.80-6.75 (m,4H), 6.08 (dd, 1H, J_(HH)=1.5 Hz, J_(HF)=15.2 Hz), 5.14, (d, 1H,J_(HH)=8.3 Hz), 4.59 (ddd, 1H, J_(HH)=3.7, 1.5 Hz, J_(HF)=51.9 Hz), 4.43(ddd, 1H, J_(HH)=7.4, 4.0 Hz, J_(HF)=19.1 Hz), 4.24-4.23 (m, 1H), 3.79(s, 6H), 3.62 (dd, 1H, J_(HH)=11.2, 2.0 Hz), 3.35 (dd, 1H, J_(HH)=11.1,2.0 Hz), 1.00 (s, 9H); ¹³C NMR (126 Hz, CDCl₃) δ 168.4, 161.8, 158.72,158.66, 148.9, 143.9, 139.4, 135.71. 135.70, 135.1, 134.8, 134.7, 132.3,132.2, 131.3, 130.4, 130.2, 130.1, 129.1, 128.2, 128.0, 127.91, 127.89,127.2, 113.19, 113.16, 102.2, 92.5 (d, JCF=194.4 Hz), 87.7 (d,J_(CF)=34.5 Hz), 87.2, 82.4, 70.0 (d, J_(CF)=15.4 Hz), 60.7, 60.4, 55.2,26.6.

Synthesis of Compound 4a

Compound 3a (14.5 g, 16.3 mmol) was dissolved into 3% trichloroaceticacid/CH₂Cl₂ solution (200 mL) containing triethylsilane (8.0 mL, 50.1mmol) and stirred for 1 hour at room temperature. After the solution waswashed by sat. aq. NaHCO₃ three times, collected organic layer was driedover MgSO₄, filtered, and evaporated. Obtained crude material waspurified by silica gel column chromatography (hexane/ethyl acetate, 4:1to 3:7) yielding compound 4a as a white foam (8.67 g, 91%); ¹H NMR (500MHz, CDCl₃) δ 7.89-7.88 (m, 2H), 7.68-7.64 (6H, m), 7.51-7.45 (m, 4H),7.42-7.38 (4H, m), 5.93 (dd, 1H, J_(HH)=2.9 Hz, J_(HF)=15.1 Hz), 5.73(d, 1H, J_(HH)=8.2 Hz), 4.74 (ddd, 1H, J_(HH)=4.1, 3.2 Hz, J_(HF)=52.2Hz), 4.31 (ddd, 1H, J_(HH)=5.8, 4.7, J_(HF)=15.4 Hz), 4.11-4.09 (m, 1H),3.82-3.79 (m, 1H), 3.39 (ddd, 1H, J_(HH)=12.1, 5.6, 1.5 Hz), 1.64 (br,1H), 1.11 (s, 9H); ¹³C NMR (126 Hz, CDCl₃) δ 168.3, 161.8, 149.0, 140.5,135.7, 135.2, 132.8, 132.3, 131.3, 130.5, 130.4, 130.3, 129.2, 128.02,127.96, 102.4, 91.8 (d, J_(CF)=91.8 Hz), 89.5 (d, J_(CF)=33.6 Hz), 69.5(d, J_(CF)=69.5 Hz), 60.3, 26.8.

Synthesis of Compound 6a

Anhydrous solution of compound 4a (6.5 g, 11.0 mmol) was added IBX (7.7g, 27.6 mmol) and stirred for 2 hours at 85° C. After cooling themixture in an ice bath, the precipitate in the solution was filtered offthrough celite. Collected eluent was evaporated, co-evaporated withanhydrous CH₃CN three times under argon atmosphere, and obtainedcompound 5a as a white foam was used without further purification. In aseparate flask, anhydrous CH₂Cl₂ (25 mL) solution containing CBr₄ (7.3g, 22.1 mmol) was added PPh₃ (11.6 g, 44.2 mmol) at 0° C. and stirredfor 0.5 h at 0° C. To this solution, anhydrous CH₂Cl₂ solution (25 mL)of compound 5a was added dropwise (10 min) at 0° C. and stirred for 2 hat 0° C. After diluting with CH₂Cl₂, the organic solution was washed byaq. sat. NH₄Cl, dried over MgSO₄, filtered, and evaporated. Obtainedmaterial was dissolved into minimum amount of diethyl ether and addeddropwise to excess diethyl ether solution under vigorously stirring at0° C. Precipitate in solution was filtered off through celite andeluents was evaporated. Obtained crude material was purified by silicagel column chromatography (hexane/ethyl acetate, 9:1 to 1:1) yieldingcompound 6a as a white foam (4.3 g, 52%). 1H NMR (500 MHz, CDCl₃) δ7.68-7.84 (m, 2H), 7.70-7.65 (m, 3H), 7.60-7.58 (m, 2H), 7.52-7.49 (m,2H), 7.42-7.36 (m, 4H), 7.31-7.28 (m, 2H), 7.09 (d, 1H, J=8.2 Hz), 6.25(d, 1H, J=8.9 Hz), 5.75 (dd, 1H, J_(HF)=8.24 Hz), 5.49 (dd, 1H,J_(HF)=21.4 Hz), 4.77 (t, 1H, J_(HH)=8.5 Hz, J_(HF)=8.5 Hz), 4.38 (dd,1H, J_(HH)=4.1 Hz, J_(HF)=52.1 Hz), 4.25 (ddd, 1H, J_(HH)=8.1, 4.9 Hz,J_(HF)=19.4 Hz), 1.10 (s, 9H); ¹³C NMR (126 Hz, CDCl₃) δ 167.9, 161.6,148.3, 141.4, 135.8, 134.7 (d, J_(C-Br)=139.0 Hz), 132.5, 132.2, 131.1,130.5, 130.3, 130.2, 129.2, 127.9, 102.7, 97.3, 93.3 (d, J_(CF)=39.1Hz), 91.5 (d, J_(CF)=190.7 Hz), 82.4, 73.9 (d, J_(CF)=16.4 Hz), 26.7.

Synthesis of Compound 7a-E and 7a-Z

Anhydrous solution of compound 6a (4.2 g, 5.66 mmol) in DMF (25 mL) wasadded dimethylphosphite (2.09 mL, 22.6 mmol) and triethylamine (1.58 mL,11.3 mmol) at 0° C., and then stirred overnight at room temperature.After the solution was diluted with ethyl acetate, the organic solutionwas washed with aq. sat. NH₄Cl and brine. Then the organic solution wasdried over MgSO₄, filtered and evaporated. Obtained crude material waspurified repeatedly by silica gel column chromatography (hexane/ethylacetate, 9:1 to 1:1) until all pure isomeric compound were collectedseparately, giving compound 7a-E (1.95 g, 52%); ¹H NMR (500 MHz, CDCl₃)δ 7.87-7.85 (m, 2H), 7.89-7.85 (m, 3H), 7.61-7.59 (m, 2H), 7.52-7.48 (m,2H), 7.45-7.32 (m, 6H), 7.08 (d, 1H, J_(HH)=8.2), 6.49 (d, 1H,J_(HH)=13.7), 5.99 (dd, 1H, J_(HH)=13.7 Hz, 8.1 Hz), 5.75 (d, 1H,J_(HH)=8.2), 5.63 (d, 1H, J_(HF)=19.8 Hz), 4.43 (dd, 1H, J_(HF)=52.6 Hz,J_(HH)=4.3 Hz), 4.42 (t, 1H, J_(HH)=8.0 Hz), 4.07 (ddd, J_(HH)=7.8, 4.7Hz, J_(HF)=19.5 Hz), 1.08 (s, 9H); ¹³C NMR (126 Hz, CDCl₃) δ 148.4,140.4, 135.8, 135.7, 135.3, 133.3, 132.3, 132.4, 132.1, 131.1, 130.5,130.4, 130.3, 129.2, 127.95, 127.93, 112.4, 102.7, 91.7 (d, J_(CF)=36.3Hz), 91.6 (d, J_(CF)=191.6 Hz), 82.8, 73.9 (d, J_(CF)=16.4 Hz), 26.7,19.1; and 7a-Z (0.58 g, 15%); 1H NMR (500 MHz, CDCl₃) δ 7.87-7.85 (m,2H), 7.68-7.65 (m, 3H), 7.61-7.59 (m, 2H), 7.52-7.48 (m, 2H), 7.42-7.39(m, 2H), 7.34-7.29 (m, 4H), 7.12 (d, 1H, J_(HH)=8.2 Hz), 6.51 (d, 1H,J_(HH)=7.4 Hz), 5.96 (dd, 1H, J_(HH)=8.4 Hz, 7.4 Hz), 5.75 (d, 1H,J_(HH)=8.2 Hz), 5.57 (dd, 1H, J_(HH)=1.2 Hz, J_(HF)=20.6 Hz), 5.04 (dd,1H, J_(HH)=8.2 Hz), 4.48 (J_(HH)=3.5 Hz, J_(HF)=53.1 Hz), 4.24 (ddd, 1H,J_(HH)=7.8, 4.9 Hz, J_(HF)=18.6 Hz), 1.09 (s, 9H); ¹³C NMR (126 Hz,CDCl₃) δ 168.0, 161.7, 148.4, 141.4, 135.9, 135.8, 135.2, 132.6, 132.5,131.2, 130.6, 130.5, 130.2, 130.1, 129.2, 127.8, 127.7, 114.5, 102.6,93.0 (d, J_(CF)=37.2 Hz), 91.6 (d, J_(CF)=191.6 Hz), 80.3, 74.3 (d,J_(CF)=16.4 Hz), 26.7, 19.1.

Synthesis of compound 9a

Anhydrous compound 7a-E (1.95 g, 2.94 mmol) and Pd(OAc)₂ (125 mg, 0.59mmol) and [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium (II)(652 mg, 1.18 mmol) were purged with argon, and then dissolved intoanhydrous THF (50 mL). After adding propylene oxide (2.06 mL, 29.4mmol), compound 8a (2.07 g, 3.24 mmol) was added in one portion andstirred at for 4 h at 70° C. After removing solvent under reducedpressure, the crude mixture was purified by silica gel columnchromatography (hexane/ethyl acetate, 50:50 to 0:100) and obtainedfractions containing compound 9a were further purified by silica gelcolumn chromatography (CH₂Cl₂-MeOH, 0% to 5%) yielding compound 9a as amixture of diastereo-isomers (2.04 g, 57%); ³¹P NMR (202 MHz, CDCl₃) δ18.3.

Synthesis of Compound 10a

Compound 9a (2.0 g, 1.64 mmol) in anhydrous THF (22.5 mL) was added 1.0M TBAF-THF (2.5 mL, 2.5 mmol) and stirred at ambient temperature for 30minutes. After diluting with CH₂Cl₂ (120 mL), the organic layer waswashed with brine, dried over MgSO₄, filtered, and then evaporated.Obtained crude material was purified by silica gel column chromatography(1% TEA-CH₂Cl₂/MeOH, 0% to 6%) yielding compound 10a (1.52 g, 94%); ³¹PNMR (202 MHz, CDCl₃) δ 19.0, 18.7.

Synthesis of Compound 11a

Compound 10a (589.7 mg, 0.6 mmol) was rendered anhydrous by repeatedco-evaporation with anhydrous CH₃CN and then dissolved into anhydrousCH₂Cl₂ (6.0 mL). To this solution N,N-diisopropylethylamine (0.31 mL,1.8 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.16mL, 0.72 mmol) were added at 0° C. After stirring for 30 min at 0° C.,the reaction mixture was diluted with excess CH₂Cl₂. The organic layerwas repeatedly washed with aq. sat. NaHCO₃, dried over MgSO₄, filtered,and evaporated. The obtained crude material was purified by silica gelcolumn chromatography (1% TEA-CH₂Cl₂/MeOH, from 100% to 4%) yieldingcompound 11a as a white foam (570 mg, 80%); 31P NMR (202 MHz, CDCl₃) δ150.3, 151.2, 151.1, 151.0, 18.72, 18.65, 18.55, 18.3.

Synthesis of Compound 4b

Anhydrous solution of compound 3b (1.35 g, 2.0 mmol) in pyridine (10 mL)was added DIPEA (0.63 mL, 3.6 nnol) and benzoyl chloride (0.35 mL, 3.0mmol), and stirred for 3 hours at room temperature. After diluting withexcess CH₂Cl₂, the organic solution was washed with aq. sat. NaHCO₃ andbrine. After drying over MgSO₄, filtered and evaporating, obtained crudematerial was used for the next reaction without further purification.Obtained crude material containing compound 3b was added 3%trichloroacetic acid in CH₂Cl₂ (25 mL) and triethylsilane (1 mL, 6.26mmol), and stirred for 1 hour at room temperature. After the reactionmixture was diluted with CH₂Cl₂, the solution was washed with sat.NaHCO₃ aq. three times, dried over MgSO₄, filtered, then evaporated.Obtained crude material was purified by silica gel column chromatography(hexane/ethyl acetate, 4:1 to 1:4) yielding pure compound 4b (596.7 mg,63% in 2 steps); ¹H NMR (500 MHz, DMSO-d6) δ 8.13 (d, 1H, J_(HH)=8.2Hz), 7.95 (d, 2H, J_(HH)=7.3 Hz), 7.81 (t, 1H, J_(HH)=7.5 Hz), 7.69-7.68(m, 2H), 7.64-7.59 (m, 4H), 7.49-7.42 (m, 6H), 5.93 (d, 1H, J_(HH)=4.6Hz), 5.26 (t, 1H, J_(HH)=4.6 Hz), 4.36 (dd, 1H, J_(HH)=4.6, 4.6 Hz),4.02-4.00 (m, 1H), 3.65-3.61 (m, 1H), 3.54 (dd, 1H, J_(HH)=4.6, 4.6 Hz),3.09 (s, 3H), 1.03 (s, 9H); ¹³C NMR (126 Hz, DMSO-d6) 169.8, 162.1,149.5, 141.3, 136.1, 135.9, 135.8, 133.4, 133.2, 131.5, 130.7, 130.52,130.48, 130.0, 128.4, 128.3, 102.1, 86.7, 85.6, 82.8, 79.7, 70.8, 60.2,57.8, 27.2, 19.4; HRMS (ESI) m/z calcd for C₃₃H₃₅N₂O₇Si⁻ [M−H]⁻ m/z599.2219, found m/z 599.2258.

Synthesis of Compound 6b

Anhydrous solution of compound 4b (300.4 mg, 0.5 mmol) in CH₃CN (5 mL)was added IBX (350 mg, 1.3 mmol) and stirred for 2 hours at 85° C. Aftercooling the solution at 0 35° C., the precipitate was filtered off bycelite-filtration. Obtained eluent containing compound 5b wasevaporated, rendered anhydrous by repeated co-evaporation with anhydrousCH₃CN, and used for the next reaction without further purification.Separatory prepared anhydrous solution of CBr₄ (331.6 mg, 1.0 mmol) inCH₂Cl₂ (5.0 mL) was added triphenylphosphine (524.6 mg, 2.0 mmol) at 0°C. in one portion and stirred at 0° C. for 30 minutes. To this solution,compound 5b in anhydrous CH₂Cl₂ (1.5 mL) was added dropwise (10 min) at0° C. and stirred for 2 h at 0° C. The solution was then diluted withCH₂Cl₂ and washed with sat. NaHCO₃ aq. and brine. After the organicsolution was dried over MgSO₄, filtered and evaporated, obtained crudematerial was purified by silica gel column chromatography (hexane/ethylacetate, 9:1 to 4:6) yielding compound 6b (210.9 mg, 56%); ¹H NMR (500MHz, CDCl₃) δ 7.88 (d, 2H, J_(HH)=7.3 Hz), 7.70-7.62 (5H, m), 7.51-7.38(m, 9H), 7.08 (d, 1H, J_(HH)=8.2 Hz), 6.26 (d, 1H, J_(HH)=8.6 Hz), 5.75(d, 1H, J_(HH)=8.2 Hz), 5.68 (d, 1H, J_(HH)=0.8 Hz), 4.84 (dd, 1H,J_(HH)=8.6 Hz, 8.6 Hz), 3.86 (dd, 1H, J_(HH)=7.5 Hz, 5.0 Hz), 3.30 (s,3H), 3.18 (br, 1H), 1.11 (s, 9H); ¹³C NMR (126 Hz, CDCl₃) 168.3, 161.7,148.6, 138.9, 135.9, 135.8, 134.3, 132.6, 132.4, 131.2, 130.5, 130.4,130.3, 129.2, 128.0, 127.9, 102.4, 97.5, 90.0, 82.44, 82.39, 74.4, 58.2,26.7, 19.1; HRMS (ESI) m/z calcd for C₃₄H₃₃Br₂N₂O₆Si⁻ [M−H]⁻ m/z751.0480 [M−H]⁻, found m/z 753.6495.

Synthesis of 7b-E and 7b-Z

Anhydrous solution of compound 6b (6.11 g, 8.1 mmol) in DMF (35 mL) wasadded dimethylphosphite (2.97 mL, 34.0 mmol) and triethylamine (2.26 mL,17.0 mmol) at 0° C., and then stirred overnight at room temperature.After the solution was diluted with ethyl acetate, the organic solutionwas washed with sat. NH₄Cl aq. and brine. Then the organic solution wasdried over MgSO₄, filtered and evaporated, and obtained crude materialwas purified repeatedly by silica gel column chromatography(hexane/ethyl acetate, 9:1 to 1:1) until all pure isomeric compound werecollected separately, giving compound 7b-E (3.0 g, 55%); ¹H NMR (500MHz, CDCl₃) δ 7.89-7.87 (m, 2H), 7.70-7.62 (m, 5H), 7.51-7.39 (m, 8H),7.10 (d, 1H, J_(HH)=8.3 Hz), 6.47 (dd, 1H, J_(HH)=13.6, 0.8 Hz), 6.01(dd, 1H, J_(HH)=13.6, 7.9 Hz), 5.76-5.74 (m, 2H), 4.51 (dd, 1H,J_(HH)=7.8, 7.8 Hz), 7.36 (dd, 1H, J_(HH)=7.8 Hz, 4.9 Hz), 3.34 (s, 3H),3.17 (dd, 1H, J_(HH)=4.7, 1.2 Hz), 1.09 (s, 9H); ¹³C NMR (126 Hz, CDCl₃)δ 168.3, 161.7, 148.7, 138.4, 135.9, 135.8, 135.3, 133.8, 132.6, 132.4,131.2, 130.5, 130.4, 130.3, 129.2, 128.0, 127.9, 112.1, 102.3, 88.9,82.8, 82.6, 77.2, 74.2, 58.1, 26.8, 19.1; and 7b-Z (1.23 g, 22%); ¹H NMR(500 MHz, CDCl₃) δ 7.89-7.87 (m, 2H), 7.72-7.70 (m, 2H), 7.68-7.63 (m,3H), 7.51-7.44 (m, 4H), 7.41-7.37 (m, 4H), 7.16 (d, 1H, J-8.2 Hz), 6.53(dd, 1H, J_(HH)=7.4, 0.6 Hz), 6.03 (dd, 1H, J_(HH)=8.5, 7.4 Hz),5.75-5.73 (m, 2H), 5.12 (t, 1H, J_(HH)=8.1 Hz), 3.93 (dd, 1H, =6.9, 5.0Hz), 3.32 (br, 1H), 3.26 (s, 3H), 1.10 (s, 9H); ¹³C NMR (126 Hz, CDCl₃)δ 168.3, 161.8, 148.7, 139.3, 135.91, 135.85, 135.22, 132.74, 132.71,131.2, 130.8, 130.5, 130.23, 130.16, 129.2, 127.78, 127.75, 114.6,102.2, 90.1, 82.4, 80.6, 77.2, 74.8, 58.1, 26.8, 19.2.

Synthesis of Compound 8b

Anhydrous5′-O-DMTr-2′-deoxy-2′-fluoro-3′-[methyl-N,N-(diisopropyl)amino]phosphor-amidite (4.26 g, 6.0 mmol) was dissolved in 0.45 M1H-tetrazole/CH₃CN solution (27 mL, 12 mmol) and stirred for 30 minutesat room temperature. To this solution, H₂O (3.6 mL) was added andstirred for 30 minutes at room temperature. After diluting with ethylacetate, the organic solution was washed with brine six times, driedover MgSO₄, filtered and then evaporated. Obtained compound 8b with aslight amount of impurity was used for the next reaction without furtherpurification; ³¹P NMR (CDCl₃, 202 MHz) δ 8.92, 8.28.

Synthesis of Compound 9b

Anhydrous compound 7b-E (2.84 g, 4.20 mmol) and Pd(OAc)₂ (188.6 mg, 0.84mmol) and [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium (II)(931.4 mg, 1.68 mmol) were purged with argon, and then dissolved intoanhydrous THF (50 mL). After adding propylene oxide (2.94 mL, 42.0mmol), compound 9b (3.16 g, 5.04 mmol) was added in one portion andstirred at for 4 hours at 70° C. After removing solvent under reducedpressure, the crude mixture was purified by silica gel columnchromatography (hexane-ethyl acetate, 50:50 to 0:100) and obtainedfractions containing compound 9b were further purified by silica gelcolumn chromatography (1% TEA-CH₂Cl₂/MeOH, 0% to 5%) yielding compound9b as a mixture of diastereoisomers (3.3 g, 64%); ³¹P NMR (202 MHz,CDCl₃) δ 19.31, 18.72.

Synthesis of Compound 10b

Compound 9b (3.3 g, 2.70 mmol) in anhydrous THF (36.5 mL) was added 1.0M TBAF-THF (4.05 mL, 4.05 mmol) and stirred at ambient temperature for30 minutes. After diluting with CH₂Cl₂ (150 mL), the organic layer waswashed with brine, dried over MgSO₄, filtered, and then evaporated.Obtained crude material was purified by silica gel column chromatography(1% TEA-CH₂Cl₂/MeOH, 0% to 8%) yielding compound 10b (1.25 g, 47%); ³¹PNMR (202 MHz, CDCl₃) δ 19.8, 19.1.

Synthesis of Compound 11b

Compound 10b (393.2 mg, 0.4 mmol) was rendered anhydrous by repeatedco-evaporation with anhydrous CH₃CN and then dissolved into anhydrousCH₂Cl₂ (4.0 mL). To this solution N,N-diisopropylethylamine (0.21 mL,1.2 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.11mL, 0.48 mmol) were added at 0° C. After stirring for 30 min at 0° C.,the reaction mixture was diluted with excess CH₂Cl₂. The organic layerwas repeatedly washed with aq. sat. NaHCO₃, dried over MgSO₄, filtered,and evaporated. The obtained crude material was purified by silica gelcolumn chromatography (1% TEA-CH₂Cl₂/MeOH, from 100% to 4%) yieldingcompound 11b as a white foam (319.6 mg, 68%); ³¹P NMR (202 MHz, CDCl₃) δ150.7, 150.4, 150.3, 19.9, 19.5, 19.4, 18.8.

Example 8: Solid Support-Mediated Synthesis of VinylPhosphonate-Modified Oligonucleotides

A representative synthesis of an oligonucleotide having a vinylphosphinate modified intersubunit linkages is illustrated in FIG. 22 .Examples of VP-modified sequences that were synthesized can be found inFIGS. 28A and 28B.

Synthesis of Inter-Nucleotide (E)-Vinyl Phosphonate Modified RNAOligonucleotides.

The synthesis RNA oligonucleotides having one vinyl phosphonate linkagewas performed on MerMade 12 automated RNA synthesizer (BioAutomation)using 0.1 M anhydrous CH₃CN solution of 2′-modified (2′-fluoro,2′-O-methyl) phosphoramidits and vinylphosphonate-linked dimerphosphoramidites. For the solid support, UnyLinker support (ChemGenes)was used. The synthesis was conducted by standard 1.0 μmol scale RNAphosphoramidite synthesis cycle, which consists of (i) detritylation,(ii) coupling, (iii) capping, and (iv) iodine oxidation.5-(Benzylthio)-1H-tetrazole in anhydrous CH₃CN was used forphosphoramidite activating reagent, and 3% dichloroacetic acid in CH₂Cl₂was used for detritylation. 16% N-methylimidazole in tetrehydrofurane(Cap A) and 80:10:10 (v/v/v) tetrhydrofurane-Ac₂O-2,6-lutidine (Cap B)were used for capping reaction. 0.02 M 12 in THF-pyridine-H₂O (7:2:1,v/v/v) was used for oxidation and 0.1 M3-[(Dimethylamino-methylidene)amino]-3H-1,2,4-dithiazole3-thione inpyridine:CH₃CN (9:1, v/v) was used for sulfurizing. For 5′-terminalphosphorylation, bis(2-cyanoethyl)-N,N-diisopropyl phosphoramidite wasused. For the 3′-cholesterol modified RNA oligonucleotide synthesis,cholesterol 3′-lcaa CPG 500 Å (ChemGenes) was used, and RNA synthesiswas conducted in the same condition as the condition used forVP-modified RNAs. After the chemical chain elongation, deprotection andcleavage from the solid support were conducted by NH₄OH-EtOH (3:1, v/v)for 48 hours at 26° C. In the case of vinyl phosphonate modified RNA,RNA on solid support was first treated with TMSBr-pyridine-CH₂Cl₂(3:1:18, v/v/v) for 1 h at ambient temperature in RNA synthesis column.Solid support was then washed by water (1 mL×3), CH₃CN (1 mL×3) andCH₂Cl₂ (1 mL×3) by flowing solution thorough synthesis column, and thendried under vacuum. After transferring the solid support to screw-cappedsample tube, base treatment by NH₄OH-EtOH (3:1, v/v) for 48 h at 26° C.was conducted. Crude RNA oligonucleotide without cholesterol conjugatewas purified by standard anion exchange HPLC, whereas RNAs withcholesterol-conjugate were purified by reversed-phase HPLC. Obtained allpurified RNAs were desalted by Sephadex G-25 (GE Healthcare) andcharacterized by electrospray ionization mass spectrometry (ESI-MS)analysis.

Example 9: Silencing Efficacy

FIGS. 23 and 24 provide visual representations of the VP-modified siRNAstudied herein. FIG. 25 exemplifies the effect that one or more vinylphosphonate modifications in an intersubunit linkage at varyingpositions on the guide strand has on silencing. As can be seen from thedata in FIG. 25 , RISC is very sensitive to VP modification, and havinga mismatch base pair at various positions can disrupt siRNA potency.

FIGS. 26, 27A, and 27B also illustrate the ability of VP-modified siRNAto silence the mutant allele. As can be seen by FIGS. 27A and 27B,adding a mismatch in the siRNA sequence could improve allelicdiscrimination without affecting mutant allele silencing. FIG. 30demonstrates that the introduction of a VP-modified linkage next to theSNP site significantly enhanced target/non-target discrimination ofSNP-selective siRNAs. Compounds containing primary (position 6) andsecondary (position 11) SNPs were synthesized with or without aVP-modification between positions 5 and 6. As can be seen in FIG. 30 ,the presence of a VP-modification had no impact on “on target” activity,but fully eliminated any detectable silencing for non-target mRNAs. Themethod for generating the data in FIGS. 25, 26, 27A, and 27B isdescribed below.

hsiRNA Passive Delivery.

Cells were plated in Dulbecco's Modified Eagle's Medium containing 6%FBS at 8,000 cells per well in 96-well cell culture plates. hsiRNAs werediluted to twice the final concentration in OptiMEM (Carlsbad, Calif.;31985-088), and 50 μL diluted hsiRNAs were added to 50 μL of cells,resulting in 3% FBS final. Cells were incubated for 72 hours at 37° C.and 5% CO₂. The maximal dose in the in vitro dose response assays was1.5 μM compound.

Method for Quantitative Analysis of Target mRNA.

mRNA was quantified from cells using the QuantiGene 2.0 assay kit(Affymetrix, QS0011). Cells were lysed in 250 μL diluted lysis mixturecomposed of one part lysis mixture (Affymetrix, 13228), two parts H₂Oand 0.167 μg/μL proteinase K (Affymetrix, QS0103) for 30 min at 55° C.Cell lysates were mixed thoroughly, and 40 μL of each lysate was addedper well of a capture plate with 20 μL diluted lysis mixture withoutproteinase K. Probe sets for human HTT and HPRT (Affymetrix; #SA-50339,SA-10030) were diluted and used according to the manufacturer'srecommended protocol. Datasets were normalized to HPRT.

Method for Creating Bar Graph.

Data were analyzed using GraphPad Prism 7 software (GraphPad Software,Inc., San Diego, Calif.). Concentration-dependent IC₅₀ curves werefitted using a log(inhibitor) versus response—variable slope (fourparameters). For each cell treatment plate, the level of knockdown ateach dose was normalized to the mean of the control group (untreatedgroup). The lower limit of the curve was set to less than 5, and theupper limit of the curve was set to greater than 95. To create the bargraph, the percent difference was calculated by subtracting the IC₅₀value for each compound from the IC₅₀ value for each correspondingcontrol compound, dividing by the IC₅₀ value for the control compound,and multiplying by 100. If the percent difference was less than −500%,the percent difference was artificially set to −500%. The lower limit ofthe graph was cut at −300%.

The contents of all cited references (including literature references,patents, patent applications, and websites) that maybe cited throughoutthis application are hereby expressly incorporated by reference in theirentirety for any purpose, as are the references cited therein. Thedisclosure will employ, unless otherwise indicated, conventionaltechniques of immunology, molecular biology and cell biology, which arewell known in the art.

The disclosure may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting of the disclosure. Scope of the disclosure is thusindicated by the appended claims rather than by the foregoingdescription, and all changes that come within the meaning and range ofequivalency of the claims are therefore intended to be embraced herein.

Example 10: Primary Screen Yields Multiple Efficacious siRNA Sequencesfor SNP rs362307 Heterozygosity

siRNAs designed to be complimentary the HTT mRNA containing analternative mutant SNP (rs362307) (FIG. 39 ) were all screened withreporter plasmids containing the target region for the SNP of interest(FIG. 40 ). HeLa cells transfected with one of two reporter plasmidswere reverse transfected with 1.5 uM hsiRNAs by passive uptake, andtreated for 72 hours. The number following SNP represents the positionof the SNP in the siRNA. It was expected that this SNP would be moredifficult to target based on the high G/C content of the region aroundit. It appears that placing the SNP in position 3 provided the most SNPdiscrimination, without losing efficacy against the mutant allele,showing that the best SNP position is sequence-specific (FIG. 41 ). Thisprimary screening process may thus be carried out for selecting the bestSNP position for any SNP.

Example 11: When Applied to SNP rs362307, a Secondary Mismatch Continuesto Improve Allelic Discrimination

As reported in FIG. 42 , primary screen of new sequences with mismatchesintroduced into all possible positions yields multiple efficacioushsiRNAs with increased SNP discrimination at position rs362307 as well.Introducing a mismatch at position 7 and 8 appeared to increaseselectivity while preserving target silencing efficacy. Other secondarymismatches provided excellent discrimination, but less activity overall.

Example 12: Measuring SNP Discrimination in Sequences Including an SNP

To measure SNP discrimination by each of the sequences disclosed inTables 5-7 (i.e., each hsiRNA having a particular SNP positionnucleotide and mismatch (MM) position nucleotide combination), psiCHECKreporter plasmids containing either a wild-type region of htt or thesame region of htt with the SNP of the sequence are prepared and testedusing a dual-luciferase. HeLa cells transfected with one of two reporterplasmids are reverse transfected with hsiRNAs by passive uptake, andtreated for 72 hours. Luciferase activities are measured in the assayswith or without the additional mismatch, and are then plotted in doseresponse curves and compared to reveal sequences yielding the bestresults in terms of discrimination and efficacy of silencing.

Example 13: Synthesis of a Phosphinate-Modified Intersubunit Linkage

A method for preparing a phosphinate-modified intersubunit linkage ofthe invention is summarized in FIGS. 44A-44C. This method involves Jonesoxidation from a free alcohol to the corresponding ketone followed by aWittig olefination to achieve the exomethylene moiety shown inintermediate compound 3. Protecting of the amide with BOM followed byhydroboration-oxidation results in the free alcohol intermediate 5.Mesylation followed by a modified Finkelstein reaction produces theiodinated intermediate 7, which then undergoes further functionalizationto achieve the methyl phosphinate monomer 9.

To achieve monomer 18, various protection and deprotection steps areemployed to achieve intermediate 13. MX oxidation produces thecorresponding ketone followed by Wittig olefination to access themethylene. Once again, hydroboration-oxidation followed by mesylationand Finkelstein reaction results in monomer 18.

Combining monomers 9 and 18 under basic conditions producesphosphinate-linked dimer 19. Acid-mediated and Pearlman's catalyzeddeprotection followed by further phosphanamine functionalization resultsin dimer 22.

The invention claimed is:
 1. A di-branched oligonucleotide compoundcomprising two RNAs, wherein the RNAs are connected to one another byone or more moieties selected from a linker, a spacer and a branchingpoint, wherein each RNA has a 5′ end, a 3′ end and a seed region,wherein each RNA is complementary to a region of a gene comprising anallelic polymorphism, and wherein each RNA comprises: a singlenucleotide polymorphism (SNP) position nucleotide at a position withinthe seed region, the SNP position nucleotide being complementary to theallelic polymorphism; and a mismatch (MM) position nucleotide located2-11 nucleotides from the SNP position nucleotide that is a mismatchwith a nucleotide in the gene.
 2. A di-branched oligonucleotide compoundcomprising two or more nucleic acid sequences, wherein the nucleic acidsequences (N) are connected to one another by one or more moietiesselected from a linker (L), a spacer (S) and optionally a branchingpoint (B), wherein each nucleic acid sequence is double-stranded andcomprises a sense strand and an antisense strand, wherein the sensestrand and the antisense strand each have a 5′ end and a 3′ end, whereinthe sense strand and the antisense strand each comprises one or morechemically-modified nucleotides, wherein each antisense strand has aseed region, wherein each antisense strand is complementary to a regionof a gene comprising an allelic polymorphism, and wherein each antisensestrand comprises: a single nucleotide polymorphism (SNP) positionnucleotide at a position within the seed region, the SNP positionnucleotide being complementary to the allelic polymorphism; and amismatch (MM) position nucleotide located 2-11 nucleotides from the SNPposition nucleotide that is a mismatch with a nucleotide in the gene. 3.The di-branched oligonucleotide compound claim 1, wherein the RNAfurther comprises at least one vinyl phosphonate modification in anintersubunit linkage having the formula:

optionally wherein a vinyl phosphonate motif is inserted next to the SNPposition nucleotide or next to the MM position nucleotide.
 4. Thedi-branched oligonucleotide compound of claim 1, wherein the di-branchedoligonucleotide compound has an hsi-RNA structure.
 5. The di-branchedoligonucleotide compound of claim 1, wherein the SNP position nucleotideis complementary to an allelic polymorphism of an htt SNP selected fromthe group consisting of rs363125, rs362273, rs362307, rs362336,rs362331, rs362272, rs362306, rs362268, rs362267, and rs363099.
 6. Thedi-branched oligonucleotide of claim 1, wherein each RNA is an ASO or adouble-stranded RNA (dsRNA).
 7. The di-branched oligonucleotide compoundof claim 1, wherein the region of a gene comprising the allelicpolymorphism comprises a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 1-10.
 8. The di-branched oligonucleotidecompound of claim 1, further comprising at least one 5′ stabilizingmoiety selected from the group consisting of phosphate, vinylphosphonate, C5-methyl (R or S or racemic), C5-methyl on vinyl, andreduced vinyl.
 9. The di-branched oligonucleotide compound of claim 1,further comprising at least one conjugate moiety selected from the groupconsisting of alkyl chain, vitamin, peptide, glycosphingolipid,polyunsaturated fatty acid, secosteroid, steroid hormone, and steroidlipid.
 10. The di-branched oligonucleotide compound of claim 2, whereinthe RNA further comprises at least one vinyl phosphonate modification inan intersubunit linkage having the formula:

optionally wherein a vinyl phosphonate motif is inserted next to the SNPposition nucleotide or next to the MM position nucleotide.
 11. Thedi-branched oligonucleotide compound of claim 2, wherein the sensestrands and the antisense strands each comprise >80% chemically-modifiednucleotides.
 12. The di-branched oligonucleotide compound of claim 2,wherein the nucleotides at positions 1 and 2 from the 5′ end of thesense and antisense strands are connected to adjacent nucleotides viaphosphorothioate linkages.
 13. The di-branched oligonucleotide compoundof claim 2, wherein each antisense strand comprises at least 15contiguous nucleotides, and wherein each sense strand comprises at least15 contiguous nucleotides and has complementarity to the antisensestrand.
 14. The di-branched oligonucleotide compound of claim 2, whereinthe compound further comprises a hydrophobic moiety attached to theterminal 5′position of the branched oligonucleotide compound.
 15. Thedi-branched oligonucleotide compound of claim 2, wherein eachdouble-stranded nucleic acid sequence is independently connected to alinker, spacer or branching point at the 3′ end or at the 5′ end of thesense strand or the antisense strand.
 16. The di-branchedoligonucleotide compound of claim 2, wherein the SNP position nucleotideis complementary to an allelic polymorphism of an htt SNP selected fromthe group consisting of rs363125, rs362273, rs362307, rs362336,rs362331, rs362272, rs362306, rs362268, rs362267, and rs363099.
 17. Thedi-branched oligonucleotide compound of claim 1, wherein the region of agene comprising the allelic polymorphism comprises a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 1-10.
 18. Thedi-branched oligonucleotide compound of claim 1, further comprising atleast one 5′ stabilizing moiety selected from the group consisting ofphosphate, vinyl phosphonate, C5-methyl (R or S or racemic), C5-methylon vinyl, and reduced vinyl.
 19. A method of inhibiting expression of agene comprising an allelic polymorphism in a cell, the method comprisingcontacting the cell with the di-branched oligonucleotide compound ofclaim
 1. 20. A method of inhibiting expression of a gene comprising anallelic polymorphism in a cell, the method comprising contacting thecell with the di-branched oligonucleotide compound of claim 2.